behavior

JoVE Behavior documents observational and experimental techniques that seek to understand human and animal behavior. Work in this field includes analysis of genetic causes and physiological responses to internal or external stimuli as well as the implicated brain regions.

Behavior

biochemistry

JoVE Biochemistry publishes experiments that investigate the structure, function, and interactions of biological molecules. The video articles in this section demonstrate methods to study nucleic acids, proteins, carbohydrates and lipids as well as enzyme function, protein folding, molecular interactions and biochemical reactions.

Biochemistry

bioengineering

JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.

Bioengineering

biology

JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.

Biology

cancer research

JoVE Cancer Research illustrates experimental approaches in biomedical research and clinical practice aimed at understanding, detecting, treating and preventing cancer. The video articles in this section demonstrate methods to study the cellular and molecular mechanisms of cancer, animal models of cancer research, development of new therapies and diagnostics, tumor imaging, chemical and radiation treatments, surgical procedures and improvements in therapy. These video articles offer researchers and clinical practitioners an efficient way to learn the technologies and experimental methods in cancer research.

Cancer Research

chemistry

JoVE Chemistry surveys a broad scope of chemical disciplines. This collection includes fundamental and applied research that investigates molecular interactions, properties, and structure spanning in the following core divisions: analytical, organic, organometallic, inorganic, bioinorganic, and biochemistry. This section also includes the design and preparation of materials and small molecules for advanced or unique applications.

Chemistry

developmental biology

JoVE Developmental Biology publishes research methodologies in the field of developmental biology. This includes methods that are used to study biological development in vitro and in vivo at the molecular, cellular, tissue, organ and whole organism levels.

Developmental Biology

engineering

JoVE Engineering encompasses a broad range of experimental and instrumental techniques utilized in physics research. Investigations in this area strive to address and answer a broad range of scientific questions, such as device mechanisms and efficiencies, using physical tools. This approach often requires a combination of specialties, and research in this area tends to be interdisciplinary with contributions from mechanical, electrical, and chemical engineers.

Engineering

environment

JoVE Environment is dedicated to research methodologies that address environmental concerns and seek to better understand Earth's ecosystem.  Special consideration is given to experimental methodologies that assess society’s impact on the environment, suggest solutions for protecting Earth’s resources, and develop sustainable fuel sources. Moving towards an environmentally conscious planet is a collaborative effort with contributions from both life and physical scientists.

Environment

genetics

JoVE Genetics publishes video articles demonstrating experimental approaches to investigate gene function and regulation. This includes methods to study gene expression, mutagenesis, epigenetics, genetic determinants of development, genetic disorders, population genetics and evolution.

Genetics

immunology and infection

JoVE Immunology and Infection includes content from a variety of fields working toward a shared initiative of improving global health. Techniques evaluating the biological response to pathogens from the molecular to organismal level are presented as well as therapeutic agents and their efficacy in treating disease.

Immunology and Infection

medicine

JoVE Medicine serves as a connection between basic research and clinical applications. Articles may present medical procedures, case studies or clinical trial methodologies. Included studies are often modeled towards predictive, preventative, personalized, and practical approaches to treatment of disease. The goal of research methodologies in this section is to improve overall patient care and prognosis.

Medicine

neuroscience

JoVE Neuroscience is a multidisciplinary section devoted to investigations of the structure, function, physiology, and pathophysiology of the brain and nervous system. Included methodologies range from molecular and cellular level studies to full central and peripheral neural systems. Potential treatment platforms and surgical techniques for neurological diseases and disorders are also presented in this section.

Neuroscience

jove journal

JoVE Journal

Scientific video journal. Peer reviewed. Multi-disciplinary. Indexed in PubMed and Web of Science.

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Research

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

Экспериментальный протокол для работы с завод-индуцированной неоднородности почвы

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

Studying Food Reward and Motivation in Humans

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as hedonics, motivation, and goal value in more objective ways. This is particularly relevant for the understanding of overeating and obesity as well as their potential treatments. In this paper are described a set of measures of food-related motivation using handgrip force as a motivational measure. These methods can be used to examine changes in food related motivation with metabolic (satiety) and pharmacological manipulations and can be used to evaluate interventions targeted at overeating and obesity. However to understand food-related decision making in the complex food environment it is essential to be able to ascertain the reward goal values that guide the decisions and behavioral choices that people make. These values are hidden but it is possible to ascertain them more objectively using metrics such as the willingness to pay and a method for this is described. Both these sets of methods provide quantitative measures of motivation and goal value that can be compared within and between individuals.

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

Изучение Продукты наградой и мотивацией у людей

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as ...

Psychosurgery

Psychosurgery, the surgical alteration or permanent removal of brain tissue to alleviate severe psychological conditions, stands as one of the most radical and controversial treatments in the history of mental health care. Its development and application have evolved significantly, marked by dramatic shifts in scientific understanding and ethical perspectives.
Historical Development of Psychosurgery
In the 1930s, Portuguese neurologist Antonio Egas Moniz introduced a surgical procedure designed to sever neural pathways between the frontal lobe and thalamus. This intervention aimed to mitigate severe mental health disorders, including schizophrenia and severe anxiety. Despite its crude methodology, the surgeries gained recognition, and Moniz was awarded the Nobel Prize in Physiology or Medicine in 1949.
Walter Freeman, an American neurologist, popularized and coined the term prefrontal lobotomy in the United States, introducing a simplified and faster technique that involved accessing the brain through the eye socket using a surgical instrument similar to an ice pick. Freeman performed over 3,000 lobotomies, often in public demonstrations, and fervently advocated for the procedure as a solution to various psychological disorders. However, his approach often overlooked long-term outcomes and ethical considerations.
Decline of Traditional Lobotomies
The widespread adoption of lobotomies from the 1930s to the 1960s resulted in significant and irreversible harm to many patients. Adverse effects included severe cognitive impairments, memory loss, emotional flatness, and personality changes. Coupled with the advent of psychiatric medications in the mid-1950s, which offered non-invasive alternatives, lobotomies fell out of favor. Reports of poor outcomes and heightened ethical scrutiny further discredited the procedure.
Modern Psychosurgical Techniques
Contemporary psychosurgery employs far more precise and controlled methods, targeting specific brain regions such as the amygdala or anterior cingulate cortex. Techniques like cingulotomy and capsulotomy are used to treat intractable psychiatric conditions such as severe obsessive-compulsive disorder (OCD) and major depressive disorder. These procedures are guided by advanced imaging technologies to minimize damage to surrounding tissues.
Stringent regulations, such as institutional review boards, now govern psychosurgery, ensuring thorough patient evaluation, informed consent, and surgeon&#39;s competency. These measures aim to balance the potential benefits of psychosurgery with its inherent risks, emphasizing its use as a last resort when all other treatments have failed.
Ethical and Scientific Considerations
Despite technological advances, psychosurgery remains a contentious field. Critics point to the procedure&#39;s historical misuse and potential for unintended consequences, while proponents argue for its value in treating otherwise untreatable conditions. Ongoing research seeks to refine surgical techniques and deepen understanding of the brain, underscoring the need for a cautious and ethical approach to this powerful intervention.

Psychosurgery involves removing or permanently destroying brain tissue to treat severe psychological conditions, making it a controversial biomedical ...

Self-Help Support Groups

Self-help support groups are voluntary, community-based organizations that provide a platform for individuals with shared concerns to exchange support, insights, and practical strategies for coping with life challenges. Typically led by group members or paraprofessionals, these groups form a cornerstone of mental health care, especially in reaching populations that are underserved by traditional healthcare systems.
Accessibility and Cost-Effectiveness
One of the primary strengths of self-help groups is their accessibility. By leveraging community resources and operating at a low cost, they serve individuals who might otherwise face barriers to mental health services, such as those with limited financial means or lower levels of education. These groups offer an inclusive space where participants can openly share experiences, seek advice, and explore solutions in a non-judgmental environment.
The Power of Shared Experience
The effectiveness of self-help groups is rooted in their emphasis on shared experience. Participants gain unique insights from peers who have faced similar challenges, fostering a sense of credibility and understanding that professionals may not always provide. For instance, a rape survivor might find meaningful support and guidance from another survivor who has successfully navigated a similar recovery process. These interactions not only validate individual experiences but also enhance emotional resilience and promote self-efficacy.
Applications in Mental Health and Beyond
Self-help groups address a wide array of issues, including substance abuse, chronic illnesses such as heart disease or cancer, and psychological trauma. Alcoholics Anonymous (AA) exemplifies the impact of these groups, with research highlighting its role in reducing alcohol consumption through mechanisms such as enhanced social networks, improved self-efficacy, increased spirituality, and alleviation of negative emotions.
The Role of Online Platforms
Advances in technology have expanded the reach of self-help groups through online platforms, offering greater accessibility for individuals unable to attend in-person meetings. However, virtual settings pose unique challenges, including the risk of misinformation. In the absence of guidance from a trained professional, members may lack the expertise and knowledge to provide optimal advice. This increases the potential for harmful group dynamics, as evidenced by certain pro-anorexia communities. Effective moderation by group leaders can mitigate these risks, ensuring a safe and constructive environment for all members.
Self-help groups exemplify the value of peer-led initiatives in mental health care, bridging gaps in accessibility and fostering collective resilience through shared understanding and mutual support.

Self-help support groups are voluntary organizations where individuals with common interests meet regularly to discuss shared concerns and provide mutual ...

Couples Therapy

Couples therapy is a therapeutic approach designed to help individuals in intimate relationships address conflicts, improve communication, and foster healthier dynamics. It is appropriate for couples at various stages, including those who are dating, married, or in long-term partnerships, and aims to support partners in navigating their unique relational challenges.
Core Principles and Techniques
Couples therapy often incorporates cognitive-behavioral principles to identify and modify negative patterns within relationships. Therapists guide partners in understanding how their individual backgrounds, beliefs, past experiences, and behaviors influence their interactions. This insight is crucial in addressing issues such as poor communication, infidelity, jealousy, sexual difficulties, substance abuse, and the complexities of blended families or two-career households.
Therapists use various techniques such as validation, reframing, structural change, and detriangulation to promote relational growth. Validation involves acknowledging each partner&#39;s emotions and beliefs, as well as fostering mutual respect and understanding. Reframing helps couples view their challenges as shared problems rather than assigning blame, encouraging collaboration. The structural change focuses on reorganizing interaction patterns to create a supportive and functional environment, while detriangulation addresses external conflicts that may be diverting attention from the relationship itself.
Navigating High-Conflict Relationships
High-conflict relationships present unique challenges due to heightened emotions and volatility. Therapists act as mediators, helping partners navigate intense disagreements and work toward constructive resolutions. These situations require a nuanced approach to de-escalate tensions and facilitate productive dialogue.
Goals and Outcomes
The primary goal of couples therapy is to equip partners with tools to enhance understanding and foster healthier interactions. For some, this process strengthens the relationship, while for others, therapy may reveal irreconcilable differences, leading to a mutual decision to separate. Regardless of the outcome, therapy emphasizes clarity and informed decision-making, enabling couples to navigate their circumstances with greater insight and confidence.
By addressing relational dynamics and promoting constructive change, couples therapy supports partners in building more harmonious connections or reaching resolutions that align with their well-being.

Couples therapy helps individuals in intimate relationships resolve conflicts and improve dynamics. Couples may be dating, married, or in long-term ...

Family Therapy

Family therapy conceptualizes psychological challenges as arising from dysfunctional interactions within the family unit, rather than as isolated issues within individuals. This approach seeks to address and transform the patterns of communication, roles, and relationships within families to promote healthier dynamics and emotional well-being for all members.
Strategic Family Therapy
Strategic family therapy emphasizes resolving communication barriers and improving problem-solving abilities among family members. Therapists employ carefully planned tasks, known as directives, to alter dysfunctional interaction patterns. One notable technique is the use of paradoxical requests, where problematic behaviors are reframed in unexpected ways to elicit change. For example, a couple prone to frequent arguments might be instructed to argue more intentionally, thereby reframing their conflict as an expression of connection. This paradoxical approach often leads to reduced conflict and an improved relationship dynamic by altering the way family members perceive and engage in their interactions.
Structural Family Therapy
Structural family therapy focuses on reorganizing the family&#39;s hierarchical structure and boundaries to enhance functionality. Therapists actively participate in family activities to observe and modify interactions, fostering healthier dynamics. For instance, helping a teenager who initially sought their family&#39;s attention by refusing to eat to express their needs verbally, ultimately leading them to stop using food refusal as a way to seek affection. This direct engagement strengthens communication, reinforces healthy boundaries, and empowers the family to support each member&#39;s needs effectively.
Effectiveness and Applications
Research demonstrates that family therapy is as effective as individual therapy, offering distinct advantages for families dealing with relational or systemic issues. By addressing problems within their broader context, family therapy promotes growth and healing not just for individuals but for the family as a cohesive unit. Approaches such as strategic and structural therapy exemplify the flexibility and efficacy of this modality, making it a valuable tool in fostering healthier familial relationships and emotional resilience.

Family therapy views psychological problems as rooted in dysfunctional family systems rather than in an individual.
The primary focus of treatment is the ...

Community Based Intervention

Community-based interventions in mental health represent a paradigm shift from institution-centered care to treatments embedded within the fabric of local communities. By prioritizing inclusion and leveraging existing societal structures, this approach fosters a supportive environment conducive to addressing mental health challenges while promoting individual dignity and agency.
Foundations of Community Mental Health Programs
Central to the success of community-based interventions is the training of local stakeholders such as teachers, nurses, and community members. These individuals are equipped with fundamental counseling skills and are often involved in workshops aimed at stress management, substance abuse reduction, and interpersonal skill development. Such initiatives help extend mental health care beyond specialized professionals, making it more accessible and reducing stigma.
The deinstitutionalization movement of the 1960s marked the beginning of this transition. It was driven by concerns over the ethical failings of institutional care, which often subjected individuals to neglect and abuse, and by economic considerations regarding the unsustainable costs of long-term institutionalization. As individuals were reintegrated into communities, the focus shifted to creating support systems that address mental health in a holistic, culturally sensitive manner.
Empowerment and Sustainability
Community-based mental health programs emphasize empowerment, enabling individuals to actively engage in their recovery and integrate fully into familial and societal contexts. This approach, championed by community psychologists, seeks to cultivate long-term well-being by embedding care within the individual&#39;s social and cultural milieu.
However, these programs require robust support in terms of local resources and government funding. Sustainable initiatives are essential to ensure continuity in care and to prevent adverse outcomes, such as homelessness, for individuals with chronic mental health conditions. Investment in community-based interventions not only alleviates the strain on formal healthcare systems but also promotes a more inclusive and equitable approach to mental healthcare.

Community-based intervention provides care within society and family, fostering inclusion and treating mental health in familiar settings like community ...

Interpersonal Psychotherapy

Interpersonal psychotherapy (IPT) is a structured, time-limited therapeutic approach initially developed to treat depression. It integrates key concepts from psychodynamic, humanistic, and cognitive-behavioral therapies, making it a uniquely eclectic framework. The therapy is rooted in the interpersonal theories of Adolph Meyer and Harry Stack Sullivan, as well as John Bowlby&#39;s attachment theory, and focuses on the interplay between interpersonal relationships and emotional well-being.
Foundations of Interpersonal Psychotherapy
The foundational premise of IPT is that interpersonal relationships significantly influence mood and psychological health. Sullivan emphasized the collaborative role of the therapist as a &#34;participant observer,&#34; working to identify and address maladaptive interpersonal patterns in the client&#39;s daily life. This focus on relationship dynamics distinguishes IPT from other therapeutic models. While it draws on psychodynamic concepts, IPT diverges from traditional psychodynamic therapy by explicitly emphasizing practical strategies to improve interpersonal functioning.
Structure and Scope of IPT
IPT is typically delivered in a short-term format, encompassing 12&#8211;16 sessions. The approach aims to enhance clients&#39; social skills, resolve interpersonal conflicts, and assist with life transitions, such as childbirth, retirement, or bereavement. The therapy is structured around specific problem areas identified during the initial assessment, such as role disputes, role transitions, unresolved grief, or interpersonal deficits. By addressing these areas, IPT helps clients develop healthier relationships and coping mechanisms.
Effectiveness Across Conditions
Originally designed to treat depression, IPT has demonstrated efficacy in combination with pharmacological treatments and as a standalone intervention. Clinical studies have shown its effectiveness in treating various conditions, including substance use disorders and eating disorders, with outcomes comparable to those achieved through cognitive-behavioral therapy. IPT&#39;s success lies in its ability to adapt its core principles to address the unique challenges associated with different mental health disorders.
The integration of theoretical insights with practical strategies makes IPT a versatile and evidence-supported approach, offering a collaborative pathway to improved interpersonal functioning and psychological health.

Interpersonal psychotherapy, or IPT, is an insight-based therapy focusing on the relationship between an individual&#39;s mood and their interpersonal ...

Electroconvulsive Therapy

Electroconvulsive therapy (ECT), or shock therapy, remains a critical biomedical intervention for severe, treatment-resistant depression. While its origins can be traced back to Hippocrates&#39; observations that malaria-induced convulsions alleviated mental illness, modern ECT has evolved significantly from its earlier, more primitive applications. First introduced in 1938 by Ugo Cerletti and his colleagues, ECT involves inducing controlled seizures using electrical currents. In its early years, patients were subjected to the procedure without anesthesia, leading to severe bodily convulsions and a barbaric reputation. Today, however, ECT is far more refined and safer due to anesthesia, muscle relaxants, and targeted stimulation.
The current ECT procedure involves administering a brief electric pulse, typically lasting less than a second, through electrodes placed on the patient&#39;s scalp. This electrical current triggers a seizure lasting approximately 60 seconds. Patients are rendered unconscious with anesthesia and muscle relaxants to prevent physical convulsions, ensuring minimal discomfort. Increasingly, the stimulation is applied to the brain&#39;s right hemisphere, reducing the risk of memory loss and cognitive impairments, which are among ECT&#39;s most debated side effects. Patients awaken shortly after the procedure with no memory of the treatment, and recovery occurs within 30 minutes.
ECT is primarily used for individuals with severe depression who do not respond to medication or psychotherapy. It is particularly effective for cases involving acute suicidality, offering rapid mood improvement. Studies report that after two to four weeks of treatments &#8212; typically three sessions per week &#8212; approximately 70% of patients experience significant relief. This success has been attributed to ECT&#39;s ability to reduce overactivity in brain regions associated with depression and potentially stimulate neurogenesis, promoting the growth of new neurons and synaptic connections.
Despite its effectiveness, ECT remains controversial due to its side effects, including memory loss and cognitive impairment. Though modern advancements, such as unilateral stimulation and refined protocols, have reduced these risks, they still warrant careful consideration. Long-term efficacy also remains uncertain, as relapses can occur, necessitating maintenance therapies like antidepressants or exercise. Nonetheless, ECT continues to save lives, especially in severe cases of depression where other treatments fail. For many, its benefits far outweigh the risks.

Electroconvulsive therapy, or ECT, commonly known as shock therapy, originated from Hippocrates&#39; observations in ancient Greece, who noted that ...

Beck's Cognitive Therapy

Cognitive therapy is a psychological approach designed to address distortions in thinking, which can lead to negative emotions and unrealistic beliefs. These cognitive distortions often influence how individuals interpret and respond to situations, exacerbating emotional distress. Below are some prevalent cognitive distortions, their characteristics, and examples of how they manifest in thought processes.
Arbitrary Inference
Arbitrary inference involves making conclusions without sufficient evidence, often based solely on personal assumptions. This type of distortion disregards facts and relies on conjecture. For example, an individual may interpret a canceled lunch appointment as evidence that their friend is seeing someone else, despite having no supporting evidence. This tendency to jump to conclusions can lead to unwarranted anxiety or mistrust.
Selective Thinking
Selective thinking occurs when a person fixates on one aspect of a situation, ignoring other relevant details that might provide balance or perspective. For instance, a student might receive praise for their work but focus entirely on a minor critique, concluding that their performance was poor. This cognitive bias limits the individual&#39;s ability to see the broader context of their achievements or the constructive nature of feedback.
Overgeneralization
Overgeneralization involves drawing broad, often exaggerated conclusions based on a single incident. For example, someone reprimanded at work might irrationally assume that their work relationships are doomed and envision a cascade of negative outcomes. This distortion perpetuates feelings of hopelessness and unwarranted despair.
Magnification and Minimization
In this distortion, individuals amplify the significance of negative events while downplaying positive ones. For example, a student who consistently excels academically might catastrophize a single mediocre grade as a sign of inevitable failure, disregarding their overall success. This imbalance skews self-assessment and fosters unwarranted negative self-perceptions.
Personalization
Personalization involves taking unwarranted responsibility for external events. For example, a person might attribute their partner&#39;s bad mood to their actions, ignoring external factors such as work-related stress. This distortion often results in misplaced guilt or self-blame.
Cognitive therapists aim to address these distortions by encouraging clients to evaluate their beliefs critically. Through structured questioning, such as examining the origin or evidence of a belief, clients are guided to identify and challenge illogical patterns. This process fosters more balanced and realistic thinking, improving emotional well-being and resilience.

Cognitive therapy, developed by Aaron T. Beck, addresses distorted thinking and unrealistic beliefs, particularly in depression.
Some of the common ...

Rational Emotive Behavior Therapy

Cognitive-behavioral therapies (CBTs) are grounded in the belief that our thoughts profoundly influence our emotions and actions. Advocates of CBT emphasize three core assumptions: first, that cognitions are identifiable and measurable; second, that they are central to psychological functioning; and third, that irrational or maladaptive beliefs can be replaced with rational and adaptive ones. This transformative approach to therapy has paved the way for specific models such as Albert Ellis&#39;s Rational Emotive Behavior Therapy (REBT), which integrates both cognitive and behavioral elements to foster mental well-being.
The ABCs of Rational Emotive Behavior Therapy
Ellis introduced REBT in the mid-1950s as a pioneering cognitive-behavioral framework. At its core lies the ABC model, which outlines the interaction between activating events (A), belief systems (B), and emotional and behavioral consequences (C). For instance, two individuals receiving the same test score may exhibit vastly different responses due to disparities in their belief systems. Rational beliefs, characterized by flexibility and self-acceptance, contrast sharply with irrational beliefs, which often involve unrealistic demands and catastrophic thinking.
To address irrational beliefs, Ellis extended the ABC model to include disputation (D), effective new beliefs (E), and fostering desired feelings and behaviors (F). Therapists employing REBT actively challenge clients&#39; maladaptive beliefs and encourage rational rethinking. Assignments or &#8220;homework&#8221; often reinforce this process, such as tasks designed to confront and disprove fears tied to irrational thoughts.
Cognitive-Behavioral Therapy: Expanding on Rationality
CBT builds upon the principles of cognitive psychology and behaviorism, focusing on present cognitive and behavioral interactions rather than past influences. It posits that psychological disorders stem from illogical thought patterns, which, when restructured into logical and rational frameworks, lead to symptom alleviation. The three overarching goals of CBT include symptom relief, equipping clients with coping strategies, and fostering a shift from self-defeating to self-enhancing thoughts.
By blending cognitive interventions with behavioral techniques, CBT offers a robust, action-oriented approach to mental health, emphasizing the transformative power of rational thinking and adaptive behavior.

In the mid-1950s, Albert Ellis developed rational emotive behavior therapy or REBT, a key example of a cognitive-behavioral approach.
He introduced the ...

Cognitive Therapy

Cognitive therapy, pioneered by Aaron T. Beck in the 1960s, is a structured approach to addressing psychological distress by focusing on the influence of thoughts on emotions and behaviors. All cognitive therapies involve the basic assumption that human beings have control over their feelings, and that how individuals feel about something depends on how they think about it. Unlike psychoanalytic methods that delve into unconscious processes or humanistic approaches emphasizing self-actualization, cognitive therapy targets distorted or dysfunctional thought patterns, aiming to restructure these to alleviate maladaptive behaviors and emotional responses.
Principles of Cognitive Therapy
Central to cognitive therapy is the concept of cognitive restructuring, which involves identifying and modifying distorted thinking patterns (cognitive distortions). For example, a client may interpret a single academic failure as indicative of global incompetence, leading to feelings of worthlessness. Through guided questioning, therapists challenge these irrational beliefs and help clients develop more accurate, rational perspectives. This active and structured process distinguishes cognitive therapy as an action-oriented approach rather than one focused on uncovering historical psychological roots.
Application to Depression and Other Disorders
Initially developed to treat depression, cognitive therapy has demonstrated effectiveness across a range of psychological disorders. Beck&#39;s work emphasized the role of self-defeating attitudes and negative automatic thoughts in perpetuating depressive states, even amidst positive life circumstances. By addressing these maladaptive cognitions, clients can reframe their interpretations of situations, reducing distress and promoting healthier emotional responses.

Cognitive therapy is a form of psychotherapy that emphasizes the role of thoughts in causing emotional distress and maladaptive behaviors.
It aims to ...

Group Therapy

Group therapy is a sociocultural approach to psychological treatment, where individuals with shared psychological challenges come together under the guidance of a mental health professional. This therapeutic modality offers unique opportunities for individuals to connect, share, and grow within the context of a supportive group. By fostering mutual understanding and collaboration, group therapy can address a range of psychological concerns effectively, often complementing or surpassing the benefits of individual therapy.
Benefits of Group Therapy
Group therapy promotes universality, helping participants realize that others share similar struggles, which can reduce feelings of isolation and shame. For instance, a child dealing with poor social skills may benefit from observing and practicing interpersonal techniques within a structured group setting. Similarly, a woman with postpartum depression might feel less guilt and more connection in a group of others facing the same challenges.
Altruism is another key feature: participants provide support, advice, and empathy to one another, which can enhance feelings of competence and efficacy. Additionally, the group environment fosters interpersonal learning, where individuals receive feedback from peers, enabling them to refine social behaviors and develop healthier interaction patterns. For those with specific needs, such as sexual abusers, group therapy offers a safe space for practicing social skills, modeling openness, and confronting harmful patterns.
Challenges and Considerations
While group therapy has significant advantages, it is not without challenges. Participants may initially feel reluctant to share personal issues with strangers, and confidentiality concerns can arise if members disclose private information outside the group. Personality conflicts and disagreements within the group can also present obstacles. Despite these challenges, effective facilitation ensures a balanced and inclusive environment where everyone benefits from the group dynamic.
Applications and Formats
Group therapy can adopt various forms, including psychodynamic, humanistic, behavioral, or cognitive approaches. Psycho-educational groups address specific topics, such as coping with a family member&#39;s illness or overcoming bullying, often within school settings. Structured groups have defined goals and activities, while unstructured groups allow members to determine the focus collaboratively. These diverse formats cater to different therapeutic needs, making group therapy an adaptable and powerful tool for psychological growth and healing.

Group therapy is a sociocultural approach to treating psychological disorders, where individuals with similar issues participate in guided sessions led by ...

Behavior Therapy

Behavior therapy incorporates diverse techniques rooted in classical conditioning principles to address maladaptive behaviors and anxiety disorders. These methods aim to reduce avoidance behaviors, foster adaptive coping mechanisms, and alter associations between stimuli and responses, making them effective in a wide range of therapeutic contexts.
Exposure therapy is a cornerstone of behavioral treatment for anxiety disorders. It involves systematic exposure to feared stimuli, either in real life, through visualization, or via virtual reality, to desensitize the individual. Gradual exposure, beginning with the least anxiety-provoking scenarios and progressing to more challenging ones, is designed to help clients build tolerance.
Systematic desensitization builds upon this by pairing exposure with relaxation techniques. For instance, someone with a fear of public speaking may practice relaxation while initially speaking to a single person and then gradually progressing to larger audiences, reducing anxiety through reciprocal inhibition.
Flooding involves directly confronting the most anxiety-inducing stimuli in a controlled setting to extinguish avoidance behaviors. Unlike graded exposure, flooding does not follow a hierarchical approach. For example, an individual with a phobia of dogs might be guided to interact with a calm and friendly dog immediately, enabling them to experience the absence of feared consequences, which helps diminish their fear response.
Aversive conditioning is employed to deter undesirable behaviors by pairing them with unpleasant experiences. For example, to address overeating, therapists may use aversive stimuli, such as a bitter taste, to create a negative association with the behavior. This method conditions the individual to avoid the maladaptive behavior due to its unpleasant consequences.
Relaxation techniques, including deep breathing and progressive muscle relaxation, are integral to these therapies. They work through reciprocal inhibition, whereby a physiological state incompatible with anxiety, such as relaxation, counteracts the anxious response. By combining relaxation with gradual or direct exposure, individuals can recondition their responses to previously feared stimuli, fostering long-term behavioral change.

In behavior therapy, a therapist employs principles of learning, such as classical and operant conditioning, to help clients change undesirable 
...

Modeling in Therapy

Modeling, a key technique in therapy, uses observational learning to help clients acquire and practice new skills by watching therapists demonstrate desired behaviors. This approach, rooted in Albert Bandura&#39;s concept of vicarious learning, plays a significant role in therapeutic interventions for various psychological conditions, including social anxiety, ADHD, and depression.
Participant Modeling
Participant modeling involves therapists demonstrating calm and effective behaviors in situations that clients find challenging. The therapist first models a constructive approach to the feared object or scenario and then guides the client through the same process step-by-step until the client gains the confidence to manage independently. This technique helps clients confront their fears in a controlled and supportive environment.
Assertion Training
Assertion training incorporates modeling to help individuals express their thoughts and feelings assertively while avoiding extremes of submissiveness and aggressiveness. This training teaches clients to advocate for themselves respectfully and effectively, ensuring that their needs and rights are recognized. The focus is on fostering assertiveness as the balance between passivity and hostility, a skill particularly valuable for those with social anxiety or interpersonal challenges.
Behavioral Rehearsal
Behavioral rehearsal, often integrated with assertion training, employs role-playing exercises to enhance the client&#39;s skills. The therapist assumes the role of a significant figure in the client&#39;s life, such as a supervisor or family member, allowing the client to practice responses in a simulated interaction. As a result, role reversal enables the therapist to model ideal verbal and nonverbal communication, illustrating not just what to say but also how to say it effectively. Feedback and coaching refine the client&#39;s performance, helping them build confidence and competence.
Applications Beyond Therapy
These techniques extend beyond therapy sessions by encouraging clients to apply learned skills in real-life situations. By practicing outside the therapeutic setting, clients consolidate their learning and improve their ability to handle social interactions. Modeling and social skills training are instrumental in managing conditions such as schizophrenia, autism spectrum disorders, and ADHD, where improved interpersonal and coping skills can significantly enhance quality of life.
Through structured observation, practice, and application, modeling serves as a powerful tool in helping clients overcome barriers and adopt constructive behaviors.

Modeling allows clients to learn by watching therapists demonstrate positive behaviors, such as maintaining eye contact during conversations.
Similarly, ...

Drug Therapy

The advent of drug therapy has profoundly shaped modern mental health care, providing targeted treatments for a range of psychological disorders. Psychotherapeutic drugs, classified into antianxiety, antidepressant, and antipsychotic medications, address symptoms across anxiety disorders, mood disorders, and schizophrenia. While these medications have transformed patient outcomes, they require careful management due to their potential side effects and limitations.
Antianxiety Medications
Antianxiety drugs, commonly known as tranquilizers, include benzodiazepines such as Xanax, Valium, and Librium. These medications alleviate anxiety by binding to neurotransmitter receptors that are hyperactive during periods of distress. Although benzodiazepines act rapidly and are effective for short-term relief, they pose risks of addiction, drowsiness, impaired coordination, and potentially harmful interactions with alcohol and other drugs. Alternatives like buspirone (BuSpar) offer less addictive solutions but may take longer to provide symptomatic relief. Caution is essential in prescribing these medications, particularly for long-term use, due to their addictive potential.
Antidepressant Drugs
Antidepressants regulate mood by influencing brain neurotransmitters, particularly serotonin and norepinephrine. Types of antidepressants such as tricyclics and tetracyclics, named for their molecular structures, have demonstrated efficacy but come with significant side effects such as dizziness, memory issues, and faintness. Monoamine oxidase (MAO) inhibitors, though effective, are less commonly used due to dietary restrictions and risks of high blood pressure. More commonly prescribed are selective serotonin reuptake inhibitors (SSRIs), which target serotonin reabsorption, offering fewer side effects and broad applications for mood, anxiety, and even chronic pain disorders. However, withdrawal from SSRIs requires careful management to avoid severe symptoms.
Mood Stabilizers Medications
Lithium remains a cornerstone treatment for bipolar disorder, effectively managing manic episodes and stabilizing mood. Its therapeutic benefits are tempered by a narrow safety margin, requiring careful blood monitoring to avoid toxicity. Side effects such as weight gain, thyroid issues, and kidney complications are common, highlighting the need for patient adherence and regular medical supervision.
Antipsychotic Medications
Antipsychotics have revolutionized schizophrenia treatment by reducing symptoms such as hallucinations and agitation. Neuroleptics, the most widely used class, modulate dopamine pathways but are associated with severe side effects, including tardive dyskinesia, a condition characterized by involuntary facial and limb movements. Newer atypical antipsychotics, such as Clozaril and Risperdal, also target serotonin and have fewer side effects, but challenges remain in patient adherence and long-term symptom management. Combining medication with psychotherapy and skill-building therapies improves outcomes, emphasizing the importance of holistic care.
Drug therapies, while pivotal in psychiatric treatment, require personalized and cautious administration. Side effects, risk of dependency, and adherence issues necessitate a balanced approach, often integrating pharmacological interventions with psychotherapy for optimal results.

The development of psychotherapeutic drugs revolutionized mental healthcare, offering effective treatments for anxiety, mood disorders, and 
...

Psychodynamic Therapy

Psychodynamic therapies emphasize the exploration of unconscious processes and early childhood experiences as fundamental contributors to psychological difficulties. These therapies, deeply rooted in Freud&#39;s psychoanalytic theory, aim to uncover and resolve unconscious conflicts, granting individuals insights that promote emotional and behavioral healing. Contemporary psychodynamic approaches have evolved, integrating a broader range of influences and methodologies while still valuing the unconscious mind and the therapeutic relationship.
Freud&#39;s psychoanalysis is the cornerstone of psychodynamic therapies. He proposed that unconscious conflicts, often originating in childhood, manifest as present-day psychological problems. Through techniques such as free association, dream analysis, and interpretation, psychoanalysts endeavor to illuminate these hidden struggles.
Free Association encourages clients to articulate thoughts without censorship, facilitating the emergence of repressed emotions or memories. Freud viewed moments of resistance &#8212; when clients hesitated or avoided certain topics &#8212; as indicators of unresolved conflicts.
Dream Analysis involves interpreting dreams&#39; manifest content (the recalled narrative) to reveal their latent content or hidden unconscious meanings. Freud believed that dreams serve as a disguised outlet for repressed wishes, often rooted in sexual or aggressive instincts.
Interpretation entails uncovering symbolic meanings in clients&#39; words and actions and offering hypotheses about unconscious motivations that drive behaviors.
Freud highlighted the importance of transference, where clients project feelings toward significant figures in their lives onto the therapist. This phenomenon provides a window into relational patterns and unresolved emotions, which therapists can help clients process. Conversely, resistance reflects unconscious defense mechanisms that hinder therapy progress. Psychoanalysts work to address and understand these resistances, aiding clients in confronting difficult truths.
Modern psychodynamic therapies have shifted focus, often prioritizing the conscious mind and current relationships over Freud&#39;s emphasis on sexual conflicts. They explore recurring life patterns, significant past events, and distressing emotions to provide insight. Therapists, influenced by figures like Heinz Kohut, emphasize empathy and healthy therapeutic relationships as a means to foster self-understanding and emotional resilience. Contemporary practices also adapt to practical constraints, favoring weekly sessions over the intensive regimens of Freud&#39;s era.
This evolution reflects an enduring commitment to exploring the depths of the human psyche while embracing contemporary perspectives on mental health.

Psychodynamic therapy explores the unconscious mind, such as repressed memories, conflicts, and early childhood experiences, to understand current ...

Operant Conditioning Intervention

Operant conditioning serves as a foundational principle in therapeutic interventions aimed at modifying maladaptive behaviors. Central to this approach is the notion that behaviors, both adaptive and maladaptive, are learned through reinforcement. By analyzing the environmental factors that reinforce problematic behaviors, clinicians can design interventions to weaken these reinforcements and replace maladaptive behaviors with healthier alternatives.
In operant conditioning, behaviors that are reinforced tend to recur, whereas those that are not reinforced eventually diminish or become extinct. For example, obsessive-compulsive disorder (OCD) behaviors, such as compulsive checking rituals, are maintained through negative reinforcement. A compulsive action, like repeatedly checking if a door is locked, alleviates anxiety and prevents anticipated negative outcomes, reinforcing the behavior. Therapy based on operant conditioning addresses this by extinguishing avoidance learning. The individual is gradually exposed to situations without performing the compulsion, demonstrating that the feared outcomes do not occur. Simultaneously, relaxation training and alternative coping mechanisms are introduced to reduce anxiety, breaking the cycle of compulsive reinforcement.
Behavioral therapies based on operant principles are particularly effective for populations with limited cognitive abilities, such as children or adults with developmental disabilities. In treating autism spectrum disorder (ASD), behavioral therapies have proven highly effective. It is used to address self-injurious behaviors, promote communication, and enhance cognitive and language development. Research indicates that intensive early interventions can significantly improve outcomes for children with ASD.
One widely implemented operant conditioning technique is the token economy. This method is commonly used in controlled environments, such as psychiatric hospitals, to encourage positive behaviors. Tokens, which serve as secondary reinforcers, are awarded for desirable actions and can be exchanged for privileges or rewards. For instance, patients may earn tokens for completing daily tasks or engaging in social interactions, which they can later trade for amenities like extra leisure time or preferred activities. The structured reinforcement system of token economies fosters sustained behavioral improvements across diverse clinical settings.

Operant conditioning suggests that behaviors are shaped by their consequences.
Behaviors followed by positive outcomes are reinforced, such as a child ...

Humanistic Therapy

Humanistic therapies emphasize personal growth, self-understanding, and the fulfillment of human potential. Rooted in the belief that individuals inherently strive toward self-actualization, these approaches encourage clients to explore their feelings and experiences in a supportive, nonjudgmental environment. Humanistic therapies differ from psychodynamic approaches by focusing on conscious experiences, present circumstances, and the potential for self-improvement rather than past conflicts and unconscious drives.
Carl Rogers&#39; client-centered therapy is a cornerstone of humanistic psychotherapy. It aims to foster self-acceptance and unity between the client&#39;s actual self and ideal self. Therapists create a therapeutic environment characterized by three essential elements:
<ol>
	<li>Unconditional Positive Regard: Therapists affirm the inherent worth of clients, offering nonjudgmental acceptance to promote self-acceptance.</li>
	<li>Empathy: By deeply understanding and reflecting on the client&#39;s emotions, therapists help clients feel heard and validated.</li>
	<li>Genuineness: Therapists are authentic and transparent, fostering a genuine connection with the client.</li>
</ol>
Active listening and reflective speech are central techniques that allow clients to identify and articulate their feelings. For example, a therapist might mirror a client&#39;s grief or anger, encouraging deeper emotional exploration.
Gestalt therapy, developed by Fritz Perls, focuses on integrating conflicting aspects of the self to achieve wholeness. Techniques like the two-chair dialogue allow clients to confront and synthesize opposing parts of their personality, fostering authenticity and emotional balance. Gestalt therapy emphasizes the &#34;here and now,&#34; urging clients to take responsibility for their feelings and actions. Techniques such as the two-chair method in Gestalt therapy, where clients alternate speaking from different perspectives to address and resolve internal conflicts, help promote self-awareness and integration.
Existential therapy, exemplified by Viktor Frankl&#39;s logotherapy, explores meaning-making as a response to life&#39;s challenges. Frankl believed that even in dire circumstances, individuals could find purpose and maintain dignity. Existential therapists help clients confront themes like freedom, isolation, and mortality, encouraging personal responsibility and resilience.
Humanistic principles are integral to various contemporary approaches, including motivational interviewing, which leverages empathy and reflective listening to encourage behavior change. Whether addressing mental health conditions or lifestyle modifications, humanistic therapies remain influential in fostering emotional awareness, self-empowerment, and authentic living.

Humanistic therapy focuses on self-healing, conscious thoughts, the present, and self-fulfillment, encouraging individuals to realize their potential and ...

Treatment Strategies for Psychological Disorders

Treatment approaches for psychological disorders fall into three main categories: psychological, biological, and sociocultural. Each approach targets different aspects of mental health, requiring varying levels of education and training.
Psychological therapies focus on modifying emotions, thoughts, and behaviors through talking, interpreting, listening, rewarding, challenging, and modeling. Clinical psychologists, counselors, and social workers commonly practice psychotherapy. Clinical psychologists typically hold a Ph.D. or Psy.D., which involves years of graduate education, supervised clinical practice, and a clinical internship. Licensing and certification are two processes through which society regulates psychotherapy practitioners. Specific laws govern the requirements for obtaining a license or certification, ensuring that professionals meet specific educational and training standards. These regulations outline the necessary qualifications and mandate formal examinations to assess the practitioner&#39;s skills and competency before allowing them to practice.
Biological therapies, or biomedical therapies, aim to treat psychological disorders by altering physical body functions, often through medications. This approach views mental health disorders similarly to physical diseases, requiring medical intervention. Practitioners of biological therapies are generally medical doctors (MDs), with psychiatrists specializing in this field. Psychiatrists undergo medical school, residency, and specialized training in psychiatry. While both psychologists and psychiatrists can administer psychotherapy, only psychiatrists can prescribe medication. If medication is necessary, a psychotherapist may refer clients to a psychiatrist for prescriptions.
Sociocultural therapies emphasize the impact of social and cultural factors on mental health. This approach considers the individual&#39;s relationships, societal roles, and cultural influences in developing and treating psychological issues. Practitioners include psychotherapists, social workers, and psychiatric social workers, who are trained to intervene in the context of these social systems. Social workers typically hold a master&#39;s degree in social work (MSW), and psychiatric social workers receive additional training in mental health. Paraprofessionals, individuals without formal mental health qualifications but trained by professionals, can also assist, often bringing personal experience with specific disorders.

Treatment strategies for psychological disorders fall under three broad approaches - psychological, biological, and sociocultural.
The psychological ...

Psychotherapy

Psychotherapy is a versatile, nonmedical approach aimed at helping individuals address emotional, behavioral, and interpersonal issues to enhance their overall well-being. It can involve one-on-one sessions, couples counseling, or small group discussions with a therapist. The therapeutic process includes various techniques such as open discussion, interpretation of thoughts and behaviors, active listening, positive reinforcement, and role modeling. Psychotherapy aims to support individuals in understanding themselves better, whether or not they have diagnosed mental health conditions.
Insight Therapies
Insight therapies focus on uncovering the psychological motives behind behavior. By exploring unconscious processes, emotions, and past experiences, these therapies aim to help individuals better understand their actions and thought patterns. Psychodynamic therapy, based on Freud&#39;s psychoanalytic principles, delves into unresolved conflicts stemming from childhood. Humanistic therapy, developed by Carl Rogers, emphasizes personal growth and self-actualization, encouraging clients to explore their potential. Existential therapy addresses questions of meaning, freedom, and responsibility, while group therapy fosters interpersonal learning through shared experiences within a group setting.
Action Therapies
Action therapies, on the other hand, focus on modifying harmful thoughts and behaviors without delving deeply into their origins. Cognitive and cognitive-behavioral therapies (CBT) are the most widely used action therapies. Cognitive therapy helps individuals identify and challenge distorted or irrational thought patterns, while CBT combines this cognitive restructuring with behavioral techniques. The aim is to replace negative thought patterns with healthier, more adaptive ones and to encourage positive behavioral changes. These therapies are highly effective in treating conditions such as depression, anxiety disorders, and phobias.

Psychotherapy is a nonmedical approach designed to help individuals recognize and address emotional, behavioral, and interpersonal problems to improve ...

Elements Crucial for Effective Psychotherapy

Research has highlighted several critical factors that influence the effectiveness of psychotherapy, such as the therapeutic alliance, the therapist, and the client.
The Therapeutic Alliance
The therapeutic alliance refers to the relationship between the therapist and the client. The alliance strengthens when the therapist and the client engage in a nurturing, supportive, trusting, empathetic, and respectful relationship, improving therapeutic outcomes. Therapists must monitor this relationship continuously, as studies suggest that clients who rate the alliance poorly are more likely to discontinue therapy or have negative experiences. Conversely, those who share a strong bond with their therapist are more likely to see successful outcomes.
The Therapist
The effectiveness of psychotherapy can depend significantly on the therapist&#39;s skill set and expertise. Experienced therapists deeply understand psychological issues and are committed to adapting their approach based on ongoing progress. Research also suggests that the compatibility between the therapist&#39;s style and the client&#39;s personality plays a crucial role in therapy success. For example, a more flexible and laid-back therapist may succeed with a client who resists change, while a directive approach may work better for others.
The Client
Another major determinant of therapeutic outcomes is the client&#39;s role in the process. Meta-analyses suggest that the client&#39;s participation is one of the most critical factors in determining success. While clients often seek therapy for life challenges, their strengths, skills, and motivation largely contribute to their progress. Research has consistently shown that therapy&#39;s success depends significantly on the client&#39;s engagement, and clients who fail to participate actively are more likely to experience unsatisfactory results.

Effective psychotherapy depends on three crucial elements&#8212; the therapeutic alliance, the therapist, and the client.
The therapeutic alliance refers ...

Conduct Disorder

Conduct disorder is a complex mental health diagnosis characterized by a repetitive and persistent pattern of behavior that violates societal norms, the rights of others, or age-appropriate rules. The diagnostic criteria for conduct disorder require the presence of at least three problematic behaviors within the past 12 months, with at least one occurring in the past six months. These behaviors are grouped into four categories: aggression toward people and animals; destruction of property; deceitfulness or theft; and serious violations of rules. The severity of symptoms, age of onset, and associated features all play crucial roles in understanding the disorder&#39;s impact and prognosis.
Diagnostic Features and Severity
The behaviors defining conduct disorder range from bullying, physical cruelty, and theft, to truancy and property destruction. Severity is categorized as mild, moderate, or severe, based on the number of symptoms and their impact on others. Childhood-onset conduct disorder, marked by symptoms before age 10, is associated with a poorer prognosis compared to adolescent-onset conduct disorder. Those with the specifier &#34;limited prosocial emotions&#34; &#8212; such as lack of remorse, callousness, or shallow affect &#8212; exhibit a more severe and persistent course.
Associated Factors and Risks
A combination of genetic, environmental, and temperamental factors influences conduct disorder. Risk factors include parental neglect, inconsistent discipline, exposure to violence, and association with delinquent peers. Neurobiological findings, such as reduced autonomic fear responses, and structural differences in brain regions associated with emotional regulation, have been observed but are not diagnostic. Co-occurring conditions, including ADHD, substance use disorders, and mood disorders, are common and can exacerbate the symptoms of conduct disorder.
Developmental Trajectory
Conduct disorder typically emerges during middle childhood or adolescence, often following oppositional defiant disorder. Symptoms evolve with age, beginning with less severe behaviors such as lying and escalating to more severe actions like theft and aggression. While the majority of cases remit by adulthood, childhood-onset conduct disorder is strongly predictive of antisocial personality disorder and other psychosocial difficulties later in life.
Sociocultural and Gender Considerations
The expression of conduct disorder behaviors differs by gender, with boys more likely to exhibit physical aggression and girls more often engaging in relational aggression or truancy. Cultural and environmental contexts, such as systemic oppression or high-crime areas, may influence the prevalence and manifestation of conduct disorder, necessitating careful diagnostic evaluation to avoid bias.
Conduct disorder presents significant challenges for affected individuals, families, and communities. Its association with academic failure, legal issues, and health risks underscores the importance of early intervention and comprehensive treatment approaches tailored to the individual&#39;s developmental and social context.

Conduct disorder involves a repetitive pattern of behavior violating others&#39; rights or societal norms, typically emerging in childhood or 
...

Intellectual Disability

Intellectual disability (ID) is a neurodevelopmental condition characterized by deficits in intellectual and adaptive functioning that manifest during the developmental period. This condition encompasses challenges in reasoning, memory, problem-solving, and learning, accompanied by impairments in everyday life skills, such as communication, self-care, and social interactions. Intellectual disability affects approximately 1% of the population in the United States, impacting an estimated 5 million individuals.
Assessment and Diagnosis
Historically, the diagnosis of intellectual disability was heavily reliant on IQ scores, with an IQ below 70 considered a diagnostic threshold. However, recent standards, such as those outlined in the DSM-5, prioritize adaptive functioning over rigid IQ thresholds. Current assessments evaluate impairments across three domains: conceptual, social, and practical skills. Conceptual abilities include literacy, numeracy, and time management, while social skills cover empathy, interpersonal communication, and compliance with social norms. Practical skills pertain to personal care, job responsibilities, and daily routines.
Adaptive functioning is evaluated in the context of the individual&#39;s life circumstances. For example, two individuals with similar IQ scores might differ significantly in functionality &#8212; one might live independently with minimal support, while the other might require institutional care. This shift in diagnostic criteria emphasizes functional capabilities rather than test scores, allowing for a more nuanced understanding of intellectual disability.
Causes and Classifications
The etiology of intellectual disability can be organic or cultural-familial. Organic causes include genetic disorders, such as Down syndrome and Fragile X syndrome, and prenatal exposure to alcohol or toxins,&#160; which result in fetal alcohol syndrome. Fetal alcohol syndrome is a condition that results from exposing a developing embryo to alcohol, and intelligence levels can range from below average to levels associated with intellectual disability. Environmental factors, such as malnutrition, lack of stimulation, and inadequate healthcare, often underlie cultural-familial intellectual disability. This type of intellectual disability, with no identifiable physical damage, typically results in milder impairments.
Classifications range from mild to profound, based on the severity of deficits. Individuals with mild ID may achieve partial independence with support, while those with profound ID often remain fully dependent on caregivers due to severe limitations in all adaptive domains.
Holistic Perspectives
While intellectual disability affects cognitive and adaptive skills, it does not diminish the individual&#39;s capacity for forming meaningful relationships and contributing to their community. Understanding and addressing their diverse needs through tailored support and interventions can significantly enhance their quality of life and social integration.

Intellectual disability, previously termed as mental retardation, has been defined by significant cognitive deficits&#8212;which have been established ...

Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS): Isolation of Splenic B Lymphocytes

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The overall function of the immune system is to defend the body against infectious organisms and other invaders. White blood cells, or leukocytes, are the key players of the immune system. Upon infection, they are activated and initiate an immune response. Leukocytes can be divided into various sub-populations (e.g., myeloid cells, lymphocytes, dendritic cells) based on different parameters that can be biological, physical, and/or functional (e.g., size, granularity, and secretion). One way to characterize leukocytes is through their surface proteins, which are mainly receptors. Each leukocyte population expresses a specific combination of receptors (e.g., cytotoxic, activating, migration receptors) that can define subsets among populations. As the immune system encompasses a wide range of cell populations, it is essential to characterize them to decipher their participation in the immune response.
Flow cytometry (FC or FCM) is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogenous cell mixture. Flow cytometers are composed of three main sub-systems: fluidics, optics, and electronics. The fluidics system transports the cells in a stream such that they pass in front of a laser one by one. The optics system consists of light sources (lasers) to illuminate the particles, optical filters to direct the resulting light, and fluorescent signals to appropriate detectors. Finally, the electronics system converts the detected light signals into electronic signals that can be processed by the computer. As an individual cell passes in front of the laser beam, it scatters light. A detector in front of the beam measures forward scatter (FS) and several detectors to the side measure side scatter (SC). The FS correlates with cell size and SC is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone.
In addition to analyzing a cell&#39;s size, shape, and complexity, flow cytometry is widely used for detecting the expression of cell surface receptors (1). This is accomplished by using fluorochrome-labelled monoclonal antibodies which bind to known cell-specific receptors. Upon excitation, these bound fluorochromes emit a light of specific wavelength, called emission wavelength, which can be detected and scored. Fluorescence measurements provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors. Hematologists were first to use FC for the therapeutic follow up of immune cell populations (2). Now, it is used for a wide range of applications such as immunophenotyping, cell viability, gene expression, cell counting, and GFP analysis.
FACS (Fluorescent Activated Cell Sorter) is a specialized type of flow cytometry, which sorts a population of cells into subpopulation using fluorescent labelling. Just like conventional flow cytometry, first FS, SC, and fluorescent data are collected. Then, the machine applies a charge (negative or positive) and an electrostatic deflection system (electromagnets) facilitates the collection of charged droplets containing cells into appropriate tubes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10494/10494fig1.jpg" /> 
Figure 1: Schematic representation of FACS. Sample (1) is aspirated in the FACS (2) and passed in front of the laser (3). Cell fluorescence is sensed by fluorescence detectors (4). Finally, cells are incorporated in droplets and the cells of interest are deflected by deflection plates (5) and collected in a collection tube (6). The remaining cells go into the trash (7). <a href="https://www.jove.com/files/ftp_upload/10494/10494fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The sorting aspect of the FACS presents many advantages. Many tests can help understand the role of specific cells in immune system, such as analyses of gene expression like RT-qPCR, cell cycle, or cytokine secretion. However, cells should be purified upstream to obtain clear and specific results. Here, FACS comes in useful and the desired cells can be sorted with great purity, yielding highly reliable and reproducible results. FACS can also be used to sort cells based on nuclear or other intracellular staining and according to the presence, absence, and density of surface receptors. FACS is now a standard technique for the purification of subpopulations of cells and has the ability sort up to four populations simultaneously.
This lab exercise demonstrates how to isolate splenic leukocytes and then how to specifically sort B lymphoid cells from the splenic leukocyte cell mixture using FACS.

Flow Cytometry and FACS: Isolation of Splenic B Lymphocytes

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur ...

Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Defence against pathogens depends on surveillance by the immune system. This system is complex and comprises many cell types, each one with specific functions. This complex composition enables immune responses to a large diversity of pathogens and injuries. Adaptive immunity allows specific responses against specific pathogens. The majority of cells responsible for this type of immunity are the lymphocytes (B cells and T cells). Usually, B cells respond to extracellular infections (such as bacterial infections), and T cells respond to intracellular infections (such as viral infections). The different types of cells in lymphocyte populations can be characterized by the combination of cell surface proteins they express and/or by a panel of secreted cytokines.
Magnetic sorting allows enrichment of targeted cell populations using magnetic properties and expression of one or several cell surface proteins (1, 2). This technique consists of three steps. First, the cells are incubated with magnetic beads that are coupled with one or several monoclonal-specific antibodies. Cells that express surface proteins that bind to these antibodies attach to the magnetic beads. Then, the targeted cell populations are captured with a magnet. To finish, the targeted cells are eluted from the magnet. At the end, two sorting products are obtained, one containing unlabeled cells and the second containing the target cells coupled with the magnetic beads. Columns can be used to improve the efficiency of magnetic sorting. In the column, a non-magnetic element lengthens the path of cell through the column. Hence, cell flow is slowed down, facilitating cell capture by the magnet.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10495/10495fig1.jpg" /> 
Figure 1: Schematic representation of magnetic separation. Thymic leukocytes are stained with anti-CD3 biotinylated antibodies. After washing, streptavidin (SAV) coupled beads specifically fix the biotin on anti-CD3 antibodies. (1) Cells are transferred in a column. (2) The magnet does not retain unlabeled cells, while CD3-positive cells remain in the column. Finally, the column is separated from the magnet and (3) CD3-positive cells are eluted in medium. <a href="https://www.jove.com/files/ftp_upload/10495/10495fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are two types of magnetic sorting (3). In positive sorting, cells of interest are captured with the magnetic beads. In negative sorting, unwanted cells are removed by capturing with the magnetic beads carrying the appropriate antibodies. This MACS technique permits good enrichment of targeted cells and improves the percentage of recovered cells from 1-20% to 60-98% in an organ. After sorting, it is necessary to verify cell purity and sorting by different methods (e.g. flow cytometry). The MACS technique is ideal to enrich a target population for other experiments such as cell culture or cell cycle analysis.
In this lab exercise, we demonstrate how to isolate thymic leukocytes and thereafter enrich thymic CD3-positive cells from the mix using magnetic cell sorting technique.

MACS: Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur ...

ELISA Assays: Indirect, Sandwich, and Competitive

Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4 
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Enzyme-linked immunosorbent assay (ELISA) is frequently used to measure the presence and/or concentration of an antigen, antibody, peptide, protein, hormone, or other biomolecule in a biological sample. It is extremely sensitive, capable of detecting low antigen concentrations. The sensitivity of ELISA is attributed to its ability to detect the interactions between a single antigen-antibody complex (1). Moreover, the inclusion of an enzyme-conjugated antigen-specific antibody permits the conversion of a colorless substrate into a chromogenic or fluorescent product that can be detected and easily quantitated by a plate reader. When compared to the values generated by titrated amounts of a known antigen of interest, the concentration of the same antigen in the experimental samples can be determined. Different ELISA protocols have been adapted to measure antigen concentrations in a variety of experimental samples, but they all have the same basic concept (2). Choosing the type of ELISA to perform, indirect, sandwich, or competitive, depends on a number of factors, including the complexity of the samples to be tested and the antigen-specific antibodies available to use. The indirect ELISA is frequently used to determine the outcome of an immunological response, such as measuring the concentration of an antibody in a sample. The sandwich ELISA is best suited for analyzing complex samples, such as tissue culture supernatants or tissue lysates, where the analyte, or antigen of interest, is part of a mixed sample. Finally, the competitive ELISA is most often used when there is only one antibody available to detect the antigen of interest. Competitive ELISAs are also useful for detecting a small antigen with only a single antibody epitope that cannot accommodate two different antibodies due to steric hinderance. The protocol will describe the basic procedures for the indirect, sandwich, and competitive ELISA assays.
The indirect ELISA assay is commonly used to measure the amount of antibodies in serum or in the supernatant of a hybridoma culture. The general procedure for the indirect ELISA assay is:
<ol>
	<li>Coat wells with antigens</li>
	<li>Add serum or hybridoma culture supernatant containing antibody (primary or 1&#176; antibody)</li>
	<li>Incubate and wash</li>
	<li>Add secondary (or 2&#176;) enzyme-conjugated antibody</li>
	<li>Incubate and wash</li>
	<li>Add substrate</li>
</ol>
The sandwich ELISA assay differs from the indirect ELISA assay in that the method does not involve coating the plates with a purified antigen. Instead, a &#34;capture&#34; antibody is used to coat the wells of the plate. The antigen is &#34;sandwiched&#34; between the capture antibody and a second &#34;detection&#34; enzyme-conjugated antibody - where both antibodies are specific for the same antigen, but at different epitopes (3). By binding to the capture antibody/antigen complex, the detection antibody remains in the plate. Either monoclonal antibodies or polyclonal antisera can be used as the capture and detection antibodies. The main advantage of the sandwich ELISA is that the sample does not have to be purified before analysis. Moreover, the assay can be quite sensitive (4). Many commercially available ELISA kits are of the sandwich variety and use tested, matched pairs of antibodies. The general procedure for the sandwich ELISA assay is:
<ol>
	<li>Coat wells with capture antibody</li>
	<li>Add test samples containing antigen</li>
	<li>Incubate and wash</li>
	<li>Add enzyme-conjugated detection antibody.</li>
	<li>Incubate and wash</li>
	<li>Add substrate</li>
</ol>
Most commercially available sandwich ELISA kits come with enzyme-conjugated detection antibodies. In cases where an enzyme-conjugated detection antibody is not available, a secondary enzyme-conjugated antibody specific for the detection antibody can be used. The enzyme on the secondary antibody performs the same role, which is to convert the colorless substrate to a chromogenic or fluorescent product. The above-mentioned secondary enzyme-conjugated antibody would more like to be used in a &#34;homemade&#34; sandwich ELISA developed by an investigator who has generated their own monoclonal antibodies, for example. One drawback to using a secondary enzyme-conjugated antibody is to be sure it only binds to the detection antibody, and not the capture antibody bound to the plate. This would result in a measurable product in all wells, regardless of the presence or absence of antigen or detection antibody.
Finally, the competitive ELISA assay is used to detect soluble antigens. It is simple to perform, but it is only suitable when the purified antigen is available in a relatively large amount. The general procedure for the competitive ELISA assay is:
<ol>
	<li>Coat wells with antigen</li>
	<li>Incubate and wash</li>
	<li>Preincubate test sample with enzyme-conjugated primary antibodies</li>
	<li>Add mixture to well</li>
	<li>Incubate and wash away any unbound enzyme-conjugated primary antibody</li>
	<li>Add substrate</li>
</ol>
The &#34;competition&#34; in this assay comes from the fact that more antigen in the test sample used in step 3 will result in less antibody available to bind to the antigen coating the well. Thus, the intensity of the chromogenic/fluorogenic product in the well at the end of the assay is inversely related to the amount of antigen present in the test sample.
A key component in any type of ELISA is the titrated standards of known concentrations that will allow the user to determine the antigen concentration present in the test samples. Typically, a series of wells are designated for creating a standard curve, where known amounts of a purified recombinant protein are added to the wells in decreasing amounts. When these wells are processed at the same time as the test samples, the user can then have a reference set of absorbance values obtained from a microplate reader for known protein concentrations to go along with the absorbance values for the test samples. The user can then calculate a standard curve to which the test samples can be compared for determining the amount of protein of interest present. The standard curve can also determine the degree of precision of the user&#39;s dilution making.
Finally, the last step in each of the ELISA types listed above calls for the addition of a substrate. The degree of conversion of the substrate to product is directly related to the amount of enzyme present in the well. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most common enzymes found conjugated to antibodies. As expected, there are a number of substrates available specific for either enzyme that produce a chromogenic or fluorescent product. Moreover, substrates are available in a range of sensitivities that can increase the overall sensitivity of the assay. The user must also take into consideration the type of instrumentation available for reading the plate at the end of the experiment when picking the type of substrate to use, along with its corresponding enzyme-conjugated antibody.
Commonly used chromogenic substrates for HRP include 2,2&#39;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) and 3,3&#39;,5,5&#39;-tetramethylbenzidine (TMB), whereas p-Nitrophenyl Phosphate (PNPP) is used for AP. ABTS and TMB produce water-soluble green and blue colored reaction products, respectively. The green ABTS product has two major absorbance peaks, 410 and 650 nm, while the blue TMB product is best detected at 370 and 652 nm. The colors of ABTS and TMB change to yellow upon the addition of an acidic stop solution, which is best read at 450 nm. Color development for ABTS is slow, while it is fast for TMB. TMB is more sensitive than ABTS, and may produce a higher background signal if the enzymatic reaction proceeds too long. PNPP produces a yellow water-soluble product after AP conversion that absorbs light at 405 nm.

Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455
...

ELISPOT Assay: Detection of IFN-&#947; Secreting Splenocytes

Source: Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
ELISPOT is a standardized, reproducible assay used to detect cellular immune responses. The assay utilizes an enzyme-linked immunosorbent assay (ELISA)- based method to detect single-cell immune responses which can be visualized by spots, hence the name ELISPOT. ELISPOT was first described in 1983, by Czerkinsky, as a method of enumerating the number of B cell hybridomas producing antigen-specific immunoglobulins (1). The same group further developed the assay to measure the frequency of cytokine producing T lymphocytes. Now ELISPOT has become a gold standard for measuring antigen-specific T cell immunity in clinical trials and vaccine candidates. For example, after vaccination or during an infection, plasma cells and memory B cells secrete antibodies that provide protection. Typically, these B cell responses are assessed by measuring serum titers of antigen-specific antibodies. However, this type of analysis, typically measured by ELISA, may not include memory B cells, which can be present even in the absence of detectable serum antibody levels. Furthermore, it has been well-established that circulating memory B cells are important for the rapid and protective antibody response observed following pathogen re-exposure, thus it is critical to be able to detect these cells. Therefore, to clearly assess antigen-specific memory B-cell responses, both ELISA and ELISPOT should be used (2).
ELISPOT assay uses a plate containing membrane-lined wells that are coated with antibodies in order to capture secreted proteins of interest. Then, the plate is loaded with cells and stimuli to induce protein production. The secreted proteins are captured by the antibodies coated on the surface. After appropriate incubation time, cells are removed and the secreted molecule is detected by using a biotinylated antibody that is specific for a different epitope, as compared to the capture antibody. Next, streptavidin peroxidase is added, followed by the addition of a substrate that allows the detection of the spots (Figure 1). The strength of this assay is that it allows one to quantitate the number of cells producing the protein of interest. Importantly, one can assess if there are changes in the total number of cells producing a specific protein or if individual cells within a population are producing more protein. Moreover, it can provide information regarding kinetics and can be used to assess overall immune activation (mitogen stimulation) relative to antigen-specific responses (antigen simulation). The ELISPOT assay will permit the detection one activated cell amongst 300,000 cells following mitogenic or antigen-specific activation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10497/10497fig1.jpg" /> 
Figure 1: ELISPOT protocol overview.
The major advantages of this assay are its- a. Simplicity- the protocol is relatively simple and straightforward. It does not require technical expertise, b. Sensitivity- it permits the detection of immune cells at the single cell level and requires very few cells compared to other methods such as flow cytometry, c. Functionality- it provides quantitative data regarding immune function.
This lab exercise demonstrates the ELISPOT protocol for detection of IFN-&#947; secreting-splenocytes, but as mentioned above this assay can also be used to assess antibody secretion by B cells (3).

ELISPOT Assay: Detection of IFN-&amp;#947; Secreting Splenocytes

Source: Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum ...

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

Source: Michael S. Lee1 and Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. The first published use of IHC was in 1941 when Albert Coons used the technique to visualize the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus (1). The name, immunohistochemistry, is derived from the roots &#34;immuno-,&#34; in reference to antibodies, and &#34;histo-,&#34; in reference to the tissue sections used in IHC. The root &#34;cyto-&#34; in immunocytochemistry highlights the key difference between ICC and IHC. Whereas IHC uses sections of whole tissue, ICC uses cells that have been isolated from tissue or grown in culture. The difference in samples used means sample preparation technically differs between IHC and ICC, but otherwise the protocols for ICC and IHC are identical and one will find the terms are frequently used interchangeably.
In both IHC and ICC, antibodies with either chemical or fluorescent tags, such as peroxidase or rhodamine, respectively, are used to visualize the distribution of any antigen of interest through specific binding of the tagged antibody to the antigen. In the case of IHC, thin slices of tissue are immobilized on a slide to maintain the structure of the tissue before being stained, allowing the visualization of antigens in the context of whole tissues (Figure 1). In the case of ICC, cells are distributed evenly on a slide before being stained, allowing the visualization of antigen distribution within individual cells but not within the structure of any specific tissue. Due to the similarities between the two protocols, this protocol will focus on IHC to address the additional complexities of sample preparation involved in IHC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10498/10498fig1.jpg" /> 
Figure 1: Outline of IHC Protocol. Visual outline of an IHC protocol for paraffin-embedded tissue dissected from a mouse. This protocol uses a biotinylated secondary antibody and strepavidin-HRP to visualize the location of antibody binding. Other options, such as fluorescently tagged antibodies, are also possible. <a href="https://www.jove.com/files/ftp_upload/10498/10498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The first major decision when performing IHC is how to prepare the tissue sections in order to maintain the structure of the tissue throughout the staining process. The two main choices are formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. There is no simple answer as to which method to use as it depends on what downstream analysis will be conducted. Formalin-fixation of paraffin embedded tissues is generally thought to better preserve tissue morphology for optimal imaging while freezing fresh tissue can preserve protein function for subsequent assays outside of IHC. In addition, fresh frozen tissue sections have been shown to be more suitable for gene expression analysis (2). A third consideration is whether or not the antibodies for your antigen of interest are suitable for either fixed or frozen tissue sections, as some antibodies have only been optimized for a specific type of section and may not work for others. Finally, one also needs to determine how long they need to store the tissue sections, as fresh frozen samples must be kept at -80&#176;C and may not last beyond one year while fixed sections can be stored for much longer at room temperature. These are a few of the major considerations for determining whether to use formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. Ultimately, if one has enough tissue, it may be best just to have some of both.
In this experiment, we set out to determine if cyclin D1 expression was increased in enlarged spleens from a spontaneous mouse model of lymphoma development. Splenic tissue samples were first isolated from either wild-type mice, transgenic mice that do not have lymphoma, or transgenic mice that have spontaneously developed lymphoma. The spleen tissue samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, stained using a mouse anti-cyclin D1 primary antibody followed by a horse anti-mouse secondary antibody, and developed using 3,3-diaminobenzidine (DAB). The sections were then counterstained in Harris Hematoxylin Solution and then the sections were imaged at 20X magnification.
Reagents
Paraffin-Embedded Sections
<ol type="a">
	<li>4% Paraformaldehyde (PFA)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100%, 95%, 80%, 75%, and 50%). Can be diluted from 100% stock using double distilled water (ddH2O)</li>
	<li>Xylene</li>
	<li>IHC compatible glass slide to ensure tissue section remains attached throughout the entire procedure. IHC compatible glass slides have a specialized coating and are readily available from multiple retailers. If performing ICC, use a chambered slide. Chambered slides allow for cells to be seeded in the chambers and placed in the incubator until cells attach to the slide and reach proper confluency, at which point the chambers can be removed and staining can proceed in the same manner as IHC.</li>
	<li>Paraffin</li>
	<li>0.3% Hydrogen peroxide (H2O2)/methanol: To prepare, add 1 mL 30% H2O2 to 99 mL methanol. Store at -20&#176;C</li>
	<li>Antigen retrieval buffer: IHC citrate buffer pH 6.0</li>
</ol>
Fresh Frozen Sections
<ol type="a">
	<li>Optimal cutting temperature (OCT) embedding compound</li>
	<li>Optimal fixative: 4% PFA or acetone that has been cooled to -20&#176;C</li>
</ol>
Staining
<ol type="a">
	<li>Blocking buffer: Should be determined by user. One example is horse serum diluted in 1X PBS</li>
	<li>Diluted primary antibody: see manufacturer specifications</li>
	<li>Diluted biotinylated secondary antibody: see manufacturer specifications</li>
	<li>Diluted avidin-Horseradish Peroxidase (HRP): Only for peroxidase visualization. See manufacturer specifications.</li>
	<li>DAB or another compatible substrate</li>
	<li>Counterstain (optional)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100% and 95%)</li>
	<li>Xylene</li>
	<li>Organo/Limonene Mount</li>
</ol>

Immunohistochemistry &amp; Immunocytochemistry: Tissue Imaging

Source: Michael S. Lee1 and Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections

Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4 
1 Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455 
2 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 
4 Center for Immunology, University of Minnesota, Minneapolis, MN 55455
Pathologic analyses of tissue sections can be used to obtain a better understanding of normal tissue structure and contribute to our understanding of mechanisms of disease. Tissue biopsies, either from patients or from experimental in vivo models, are often preserved by fixing in formalin or paraformaldehyde and embedding in paraffin wax. This allows for long-term storage and for the tissues to be sectioned. Tissues are cut into thin (5 &#181;m) sections using a microtome and the sections are adhered to glass slides. The tissues sections can be stained with antibodies, which allow for the detection of specific proteins within the tissue sections. Staining with antibodies conjugated to fluorophores (also known as fluorochromes) - compounds that emit light at specific wavelengths when excited by a laser - is known as immunofluorescence. The ability to detect proteins within a section can provide information such as cell type heterogeneity within the tissue, activation of specific signaling pathways, and expression of biomarkers. Depending on the fluorophores used and type of microscope available for analysis, multiple colors can be used, which allows for multiplexed analysis of targets.
The following protocol outlines the basic steps involved in immunofluorescence staining of paraffin embedded tissue sections. It is important to note that this protocol will not include any details on the fixation of tissue, process of paraffin embedding, or sectioning of the tissues. Once tissues have been sectioned and placed on glass slides, they are rehydrated through a series of graded ethanol (EtOH) incubations. The sections are incubated with a blocking reagent to reduce non-specific binding of antibody to the tissue section. The sections are then incubated with a primary antibody that may or may not be directly labeled with a fluorophore. If the primary antibody is not directly labeled, the sections are then incubated with a secondary antibody labeled with a fluorophore. Different antibodies may require different staining conditions, thus suggestions for optimization of antibodies are included. Following washing to remove all unbound antibody, the slides are mounted with media containing DAPI to fluorescently label the nucleus. Once the mounting media has dried, the slides can be imaged using a microscope with lasers that can detect the different fluorophores.

Immunofluorescence Staining and Microscopy

Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4
1 Department of Lab Medicine and Pathology, University of Minnesota, ...

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201 
2 Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Confocal fluorescence microscopy is an imaging technique that enables increased optical resolution as compared to conventional &#34;wide-field&#34; epifluorescence microscopy. Confocal microscopes are able to achieve improved x-y optical resolution through &#34;laser scanning&#34;- typically a set of voltage-controlled mirrors (galvanometer or &#34;galvo&#34; mirrors) that direct laser-illumination to each pixel of the specimen at a time. More importantly, confocal microscopes achieve superior z-axial resolution by using a pinhole to remove out of focus light originating from locations that are not in the z-plane being scanned, thus enabling the detector to collect data from a specified z-plane. Because of the high z-resolution achievable in confocal microscopy, it is possible to collect images from a series of z-planes (also-called z-stack) and construct a 3D image through software.
Before discussing the mechanism of a confocal microscope, it is important to consider how a sample interacts with light. Light is composed of photons, packets of electromagnetic energy. A photon impinging on a biological sample can interact with the molecules comprising the sample in one of four ways: 1) the photon does not interact and passes through the sample; 2) the photon is reflected/scattered; 3) the photon is absorbed by a molecule and the absorbed energy is released as heat through processes collectively known as nonradiative decay; and 4) the photon is absorbed and the energy is then rapidly reemitted as a secondary photon through the process known as fluorescence. A molecule whose structure permits fluorescence emission is called a fluorophore. Most biological samples contain negligible endogenous fluorophores; therefore exogenous fluorophores must be used to highlight features of interest in the sample. During fluorescence microscopy, the sample is illuminated with light of the appropriate wavelength for absorption by the fluorophore. Upon absorbing a photon, a fluorophore is said to be &#34;excited&#34; and the process of absorption is referred to as &#34;excitation&#34;. When a fluorophore gives up energy in the form of a photon, the process is known as &#34;emission&#34;, and the emitted photon is called fluorescence.
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a &#34;focal point&#34; where it is maximally focused. Beyond the focal point the light again diverges. The entering and exiting beams may be visualized as a pair of cones touching at the focal point (see Figure 1, left panel). The phenomenon of diffraction imposes a limit on how tightly a beam of light can be focused - the beam actually focuses to a spot of finite size. Two factors determine the size of the focal spot: 1) the wavelength of the light, and 2) the light-gathering ability of the objective lens, which is characterized by its numerical aperture (NA). The focal &#34;spot&#34; extends not only in the x-y plane, but also in the z direction, and is in reality a focal volume. The dimensions of this focal volume define the maximum resolution achievable by optical imaging. Although the number of photons is greatest within the focal volume, the conical light paths above and below the focus also contain a lower density of photons. Any fluorophore in the light path can thus be excited. In conventional (wide-field) epifluorescence microscopy, emission from fluorophores above and below the focal plane contribute out-of-focus fluorescence (a &#34;hazy background&#34;), which reduces image resolution and contrast, as demonstrated in Figure 1, with the red cube representing fluorophore emission above the focal plane (red star) that results in out-of-focus fluorescence (top right). This problem is ameliorated in confocal microscopy, due to the utilization of a pinhole. (Figure 2, bottom right). As depicted in Figure 3, the pinhole allows emissions originating from the focal place to reach the detector (left), while blocking the out-of-focus fluorescence (right) from reaching the detector, thus improving both resolution and contrast.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig1.jpg" /> 
Figure 1. Optical resolution of epifluorescence versus confocal microscopy. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a focal volume and then diverges (left). The red star represents the focal plane of a sample that is being imaged while the red square represents fluorophore emission above the focal plane. When capturing an image of this sample using an epifluorescent microscope, the emission from the out-of-focus red square will visible and contribute to a &#34;hazy background&#34; (top right). Confocal microscopes have a pinhole which prevents the detection of light emitted outside of the focal plane, eliminating the &#34;hazy background&#34; (bottom right).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig2.jpg" /> 
Figure 2. Pinhole effect in confocal microscopy. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the highest intensity of the excitation light is at the focal point of the lens (left, red oval), other parts of the sample not in the focal point (right, red star) will get light and fluoresce. To prevent light emitted from these out-of-focus regions to reach the detector, a screen with a pinhole is present in front of the detector. Only the in-focus light (left) emitting from the focal plane is able to travel through the pinhole and reach the detector. The out-of-focus light (right) is blocked with the pinhole and fails to reach the detector.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig3.jpg" /> 
Figure 3. Principal components of a confocal laser scanning microscope. <a href="https://www.jove.com/files/ftp_upload/10501/10501fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the sake of simplicity, the mechanistic description of a confocal microscope will be limited to that of the Nikon Eclipse Ti A1R. Although there may be minor technical differences between different confocal microscopes, the A1R serves well as a good model for describing confocal microscope function. The excitation light beam, produced by an array of diode lasers, is reflected by the primary dichroic mirror into the objective, which focuses the light onto the specimen being imaged. The primary dichroic mirror selectively reflects the excitation light while allowing light at other wavelengths to pass through. The light then encounters the scanning mirrors that sweep the light beam across the specimen in an x-y manner, illuminating a single (x,y) pixel at a time. Fluorescence emitted by fluorophores at the illuminated pixel is collected by the objective lens and passes through the primary dichroic mirror to reach an array of photomultiplier tubes (PMTs). Secondary dichroic mirrors direct the emission light to the appropriate PMT. Excitation light scattered by the sample back into the objective is reflected by the primary dichroic mirror back towards the specimen, and thus prevented from entering the detection light path and reaching the PMTs (see Figure 3). This allows the relatively feeble fluorescence to be quantified without contamination by light scattered from the excitation light beam, which is typically orders of magnitude more intense than the fluorescence. Because the pinhole blocks light from outside of the focal volume, the light arriving at the detector comes from a narrow, selected z-plane. Therefore, images can be collected from a series of adjacent z-planes; this series of images is often referred to as a &#39;z-stack&#39;. By using the appropriate software, a z-stack can be processed to generate a 3D image of the specimen. A particular advantage of confocal microscopy is the ability to distinguish the subcellular localization of staining. For example, the differentiation between membrane staining from intracellular staining, which is very challenging with conventional epifluorescence microscopy (1, 2, 3).
Sample preparation is an important facet of confocal imaging. A strength of optical microscopy techniques is the flexibility to image live or fixed cells. When attempting to produce 3D images, because of the number of images that must be acquired for a z-stack, the difficulty of maintaining cell health, and the movement of live cells and their organelles, the use of fixed cells is typical. The procedure for fixing and staining cells for confocal fluorescence is similar to that conventionally used in immunofluorescence. After culture in chamber slides or on coverslips, cells are fixed using paraformaldehyde to preserve cellular morphology. Non-specific antibody binding is blocked using bovine serum albumin, milk, or normal serum. In order to maintain the specificity of the secondary antibodies, the solution used should not originate from the same species in which the primary antibodies were generated. The cells are incubated with primary antibodies that bind the antigen of interest. When labeling several cellular targets, the primary antibodies must each be derived from a different species. Antibodies tagging an antigen are then bound by fluorophore-conjugated secondary antibodies. Fluorophore-conjugated secondary antibodies should be selected so that they are compatible with the wavelengths of laser excitation available in the confocal microscope. When visualizing multiple antigens, the excitation/emission spectra of the fluorophores should differ enough so that their signals can be discriminated by microscopic analysis. The stained specimen is then mounted on a slide for imaging. A mounting medium is used to prevent photobleaching and specimen dehydration. If desired, a mounting medium containing a nuclear counterstain (e.g. DAPI or Hoechst) can be used (4).
In the following protocol, mouse fibroblasts transfected to express CD1d (LCD1) were stained with antibodies recognizing CD1d and CD107a (LAMP-1). CD1d is a major histocompatibility complex 1 (MHC 1)-like receptor present on the surface of antigen presenting cells that presents lipid antigens. LAMP-1 (lysosomal associated membrane protein-1) is a transmembrane protein primarily present in lysosomal membranes. For proper antigen presentation, CD1d is trafficked through the low pH lysosomal compartment, so LAMP-1 is being used as a marker of the lysosomal compartment for this protocol. By probing the LCD1 cells with anti-CD1d and anti- LAMP-1 that were produced in different species, secondary antibodies with unique fluorophores can be used to determine the localization of each protein in the cell and whether CD1d is present in the LAMP-1 positive lysosomal compartments.

Confocal Fluorescence Microscopy and Localization of Proteins

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Adoptive cell transfer is a method for introducing cells into a patient or study organism in order to treat a disease or study a biological process, such as haematopoiesis. Aims of adoptive transfer are various; it can be used in fundamental biology as well as in medical sciences (1, 2). In mouse models, migration and distribution of transferred cells can be studied and followed by a tracking system (cell surface marker, staining by CFSE, etc.). In cancer studies on mouse models, transfer of specific cell populations can be used as experimental treatment against tumors. Another example for this technique is creation of chimeric mice by transfer of bone marrow cells to irradiated mice or mice with a severe immunodeficiency phenotype. This mouse model can be used to assess the impact of gene deletion on a specific cell population for instance. Transfer of bone borrow cells is also used in human medical treatment. When patients are irradiated in case of cancer therapy, adoptive transfer of bone marrow allows immune system reconstitution.
The first step in this technique is to obtain the cell population of interest. The technique chosen to isolate this population depends on the level of specificity of the targeted population. The largest level of selection is the whole organ, in which all cell populations present in the organ are taken. A more precise method is selection of a target cell population, often selected by one cell surface marker. The ideal method to sort cells in this case is by magnetic sorting. Finally, the most stringent level is the selection of cells by several cell surface markers to sort very specific cell populations. Flow cytometry sorting is the most popular method for this level of selection. Once population of interest is obtained, it can be transferred to the host. Before adoptive transfer it is essential to ensure compatibility between host and donor. Indeed, regardless of transfer goal, compatibility is crucial to assure cells adoption by the host without cells rejection.
In this lab exercise, we demonstrate the adoptive cell transfer technique by transferring splenocytes from a CD45.2 mouse into a CD45.1 Rag&#947;c mouse (lacking lymphocytes) and four days later confirm the splenocytes transfer using flow cytometry (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10504/10504fig1.jpg" /> 
Figure 1: Schematic representation of adoptive transfer. (1) Splenocytes are isolated from CD45.2 mice and (2) transferred in CD45.1 Rag&#947;c mouse, control mouse is injected with PBS only. (3) 4 days after adoptive transfer, splenocytes are recovered from mice and (4) analyzed by flow cytometry. <a href="https://www.jove.com/files/ftp_upload/10504/10504fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Adoptive Cell Transfer Technique

Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
One of the main functions of the cells of the immune system is to remove target cells that have been infected with viruses or have undergone transformation into a tumor cell. In vitro assays for measuring the cytotoxic capacity of immune cells have been a staple in laboratories for many years. These assays have been used to determine the ability of T cells, NK cells, or any other immune cell to kill target cells in an antigen-specific or -nonspecific manner. Death ligands (e.g., Fas ligand or TRAIL), cytokines (e.g., IFNg or TNF), or cytotoxic granules (i.e., perforin/granzyme B) expressed by effector cells are some ways in which target cell death can be induced. With the explosion in tumor immunotherapy research in recent years, there is growing interest in finding agents to increase the cytotoxic activity of immune cells to improve patient outcomes. Conversely, some diseases are hallmarked by the overexuberant activity of immune cell cytotoxic activity, resulting in efforts to identify agents to temper these responses. Thus, having an assay in which the user can easily integrate any number of different effector cells, target cells, and/or response modifiers into the experimental design can serve as a valuable means of quickly assessing the cytotoxic capacity of effector cells and/or the responsiveness of the target cell.
These in vitro assays involve the mixing of different cell populations, as well as using a relatively low number of both effector and target cells. Thus, one necessity of the assay is to label the target cells in a manner that can easily be detected and quantitated, allowing the user to then determine the &#39;percent specific lysis&#39; mediated by the effector cells. Radioactivity - especially, chromium 51 (51Cr) in the form of Na251CrO4- is an inexpensive way to rapidly and nonspecifically label cellular proteins within the target cells (1). The short labeling and total assay times reduces the potential for significant changes in the number and/or phenotype of the target cells, which could influence the outcome of the assay. Upon the loss of membrane integrity of the target cells as a result of the cytotoxic activity of the effector cells, the 51Cr-labelled cellular proteins within the target cells are released into the culture supernatant, becoming available for quantitation. As with any assay examining the function of immune cells in vitro, there are a number of important considerations to consider improving the performance of the experiment. One of the most critical features is to use healthy effector (for maximal cytotoxic activity) and target (for maximal responsiveness and minimal spontaneous death/51Cr release) cells. Effector and target cell contact is required (leading to the common use of round-bottom 96-well plates to encourage cell-cell contact) (2). Finally, data analysis is dependent on the inclusion of positive and negative control target cell populations.
The following protocol will outline the steps for performing a standard 51Cr release assay to measure the cytotoxic ability of a population of effector cells, though a nonradioactive version using Europium has recently been developed. 51Cr is a powerful &#947;-radiation emitter. Consequently, the use of this assay requires proper radiation safety training, dedicated laboratory space, a gamma counter, and disposal of radioactive samples.
The general sequence of events in this assay are: 1) prepare 51Cr-labeled targets; 2) prepare effector cells and add to plate while target cells are labeling; 3) add labeled targets to plate; 4) incubate plate; 5) harvest supernatants; and 6) analyze data after running samples on counter. Samples are commonly prepared in triplicate, and then averaged to account for any subtle pipetting differences.
Proper PPE is important for this assay. Specifically, the user should wear a lab coat and gloves. Safety glasses may be required based on the laboratory or institution. There should be ample lead shielding for safe storage and use of the 51Cr during all steps. Finally, there should be dedicated lab space and equipment set aside for using 51Cr, including all the proper signage to indicate where samples with 51Cr are being kept and a Geiger counter equipped with gamma probe to survey the space for possible contamination.
In this lab exercise, we will determine the ability human peripheral blood mononuclear cells (PBMCs), (CpG stimulated vs. unstimulated) to kill melanoma cells, using human melanoma cell line WM793 as model and the chromium release assay.

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