وقد تم تمويل هذه الدراسة من قبل جمعية الألمانية للبحوث إلى SW (DFG WA 2804) ومنحة الجامعة لSW من مركز البحوث التجريبية والسريرية، وبالتعاون مع مركز ماكس للطب الجزيئي وشاريتيه في برلين كلية الطب ديلبروك. كان الممولين أي دور في تصميم الدراسة وجمع البيانات وتحليلها، قرار نشر أو إعداد المخطوطة. نشكر السيد روبرت يستفال للحصول على الدعم الفني خلال التدريب له في مختبرنا.

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

هذه الطريقة في توظيف 19 F / 1 H التصوير بالرنين المغناطيسي لمتابعة حركة العاصمة إلى العقدة الليمفاوية يعطي الفرصة لدراسة أنماط هجرة الخلايا المناعية في الجسم الحي. الخلايا الجذعية هي أمثلة ممتازة للهجرة بسرعة الخلايا المناعية التي تكون قادرة على المنا...

تتبع من الخلايا في الجسم الحي هو جانب هام في عدة مجالات الطب الحيوي. لهذا، وتقنيات التصوير موسع التي يمكن توطين بشكل انتقائي خلايا على مدى فترة من الوقت هي قيمة للغاية. قبل وضع ثلاثي الأبعاد التصوير بالرنين المغناطيسي (MRI)، تتبع هجرة الخلايا المناعية اقتصر على التحليلات المجهرية أو الخزعات النسيجية. وقد اكتسب تتبع الخلية بمساعدة التصوير بالرنين المغناطيسي الاهتمام هائلة في السنوات القليلة الماضية، ليس فقط بالنسبة للمناعة دراسة سلوك الخلايا المناعية في الجسم الحي، ولكن أيضا للباحثين الخلية الجذعية السريرية و. خلال منتصف 90s، أولى الدراسات على أكسيد الحديد النانوية 1 شرعت في سلسلة من التطورات لخلايا تتبع مع ​​التصوير بالرنين المغناطيسي. جسيمات أكسيد الحديد تقصير وقت الاسترخاء MR (T2 *) من الخلايا المسمى وبالتالي تسبب نضوب إشارة في صور الرنين المغناطيسي. وقد استخدمت جسيمات أكسيد الحديد لالضامة التسمية 2، ص دبقية قليلة التغصنrogenitors 3 والعديد من أنواع الخلايا الأخرى. كما تم الموافقة سريريا بعض هذه الجسيمات من قبل ادارة الاغذية والعقاقير لوصفها اللقاحات الخلوية في مرضى سرطان الجلد 4. منذ ذلك الحين في الجسم الحي أو وضع العلامات فيفو السابقين الخلايا مع جزيئات أكسيد الحديد تعتمد على تقصير من * إشارة T2 ويمكن رفع هذا الأخير أيضا حول من قبل في الجسم الحي المتصلة قابلية T2 * تأثيرات مثل ينزف الصغرى، رواسب الحديد أو فقاعات الهواء، قد يكون من الصعب تحديد الخلايا المسمى في الجسم الحي من خلفية أخرى T2 * انقراض إشارة 5. في هذه المقالة، ونحن تصف تقنية لتتبع الخلايا الجذعية (DC) في الجسم الحي من خلال توظيف 19 F / 1 H التصوير بالرنين المغناطيسي (MRI). وقدم هذه التكنولوجيا تتبع الخلية فقط في عام 2005 6، وبعد عدة سنوات من تطبيقات أقر لأول مرة لمدة 19 F في التصوير بالرنين المغناطيسي قد تم الإبلاغ عن 7. واحدة أدفا هامةntage من 19 F أكثر من أكسيد الحديد الجسيمات وسم الخلية هو وقوع البيولوجي منخفض من 19 F في الأنسجة، وهذا يجعل من الممكن لتعقب الخلايا بشكل انتقائي جدا مع الصور مجانا خلفية أساسا. وعلاوة على ذلك، فمن الممكن أن تراكب 19 F MR إشارة من الخلايا المسمى المزروعة مع الصور التشريحية التقليدية 1H تم الحصول عليها من التصوير بالرنين المغناطيسي. 19 F / 1 H التصوير بالرنين المغناطيسي هو بالتالي ذات الصلة إلى حد كبير لدراسات الهجرة التحقيق خلية في الجسم الحي. وصفت الخلايا درس مع هذا الأسلوب مع 19 الجزيئات F الغنية. المشبعة صناعيا المستمدة بالفلور التي تتكون أساسا من الكربون وذرات الفلور وتستخدم عادة لتحضير جسيمات. هذه المركبات هي غير قابلة للذوبان في الماء، وتحتاج إلى مستحلب قبل تطبيق في المختبر أو في الجسم الحي. حجم المعتاد للجسيمات PFC التي استخدمت من قبل مجموعات أخرى لفي الجسم الحي 19 التجارب تتبع F-MRIيتراوح بين 100 نانومتر و 245 نانومتر 6،8-10. ومع ذلك أظهرنا أن كفاءة في وسم الخلايا الجذعية مع perfluoro-15-تاج-5-الأثير (PFCE) زيادات جزيئات مع زيادة حجم الجسيمات (&gt; 560 نانومتر). 11

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>C57BL/6 mice</td>
 <td>Charles River, Berlin</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Gibco</td>
 <td>21875-091</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS Superior</td>
 <td>Biochrom AG</td>
 <td>S 0615</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Gibco-Invitrogen</td>
 <td>15630-056</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030-024</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Sigma Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>PFA</td>
 <td>Santa Cruz</td>
 <td>sc-281692</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfluoro-15-crown-5-ether </td>
 <td>ChemPur</td>
 <td>391-1996</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F-68 </td>
 <td>Sigma Aldrich</td>
 <td>P5556</td>
 <td></td>
 </tr>
 <tr>
 <td>Petri dishes (35 x 10 mm)</td>
 <td>VWR, Germany</td>
 <td>391-1996</td>
 <td></td>
 </tr>
 <tr>
 <td>27 &frac12; G syringes</td>
 <td>VWR, Germany</td>
 <td>612-0151</td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon cell strainers (100 &mu;m mesh) </td>
 <td>VWR, Germany</td>
 <td>734-0004</td>
 <td></td>
 </tr>
 <tr>
 <td>NMR tubes </td>
 <td>VWR, Germany</td>
 <td>634-0461</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Dissection tools</td>
 <td>FST</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CO2 incubator </td>
 <td>Binder</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Small animal MR system</td>
 <td>Bruker Biospin</td>
 <td></td>
 <td>9.4T BioSpec 94/20 USR, ParaVision Acquisition and Processing Software</td>
 </tr>
 <tr>
 <td>1H/19F dual-tunable volume RF coil </td>
 <td>Rapid Biomed, W&uuml;rzburg, Germany</td>
 <td></td>
 <td>35 mm inner diameter, 50 mm length</td>
 </tr>
 <tr>
 <td>19F spectroscopy coil </td>
 <td>in-house</td>
 <td></td>
 <td>tune/match loop coil, 4 turns, inner diameter 5 mm, 10 mm long, two capacitors for tuning and matching</td>
 </tr>
 <tr>
 <td>Isoflurane inhalation system</td>
 <td>F&ouml;hr Medical Instruments GmbH</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Animal monitoring system Model 1025</td>
 <td>SA Instruments Inc., New York, USA</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

monitoring dendritic cell migration using 19f / 1h magnetic resonance imaging

التقدم المستمر في طرائق التصوير موسع مثل التصوير بالرنين المغناطيسي (MRI) قد تحسنت بشكل كبير قدرتنا على دراسة العمليات الفيزيولوجية المرضية أو في الكائنات الحية. التصوير بالرنين المغناطيسي يثبت أيضا أن يكون أداة قيمة لالتقاط الخلايا التي تم زرعها في الجسم الحي. استراتيجيات التوسيم الخلية الأولي للتصوير بالرنين المغناطيسي استخدام مصنوعة من عوامل التباين التي تؤثر في اوقات الاسترخاء MR (T1، T2، T2 *) ويؤدي إلى تعزيز (T1) أو نضوب (T2 *) من إشارة حيث الخلايا المسمى موجودة. T2 * وكلاء تعزيز مثل الصغر وكلاء أكسيد الحديد (USPIO) وقد استخدمت لدراسة الهجرة الخلية والبعض أيضا قد وافقت عليه ادارة الاغذية والعقاقير لالتطبيق السريري. والعيب من T2 * وكلاء هو صعوبة التمييز بين الانقراض إشارة إنشاؤها بواسطة الخلايا المسمى من التحف الأخرى مثل جلطات الدم، نزيف الدقيقة أو فقاعات الهواء. في هذه المقالة، ونحن تصف تقنية ناشئة لخلايا الجسم الحي تتبع في ذلكويستند على وصفها الخلايا مع الفلور (F 19) الغنية الجسيمات. وتعد هذه الجسيمات عن طريق الاستحلاب المشبعة (PFC) مركبات وتستخدم بعد ذلك إلى خلايا التسمية، التي لاحقا يمكن تصوير بنسبة 19 F التصوير بالرنين المغناطيسي. مزايا هامة من الهيدروكربون المشبع بالفلور لتتبع خلية في الجسم الحي ما يلي: (ط) عدم وجود الكربون محدد 19 F في الجسم الحي والتي ينتج عنها بعد ذلك خالية من الصور الخلفية وكاملة خلية selectivityand (II) إمكانية لقياس إشارة الخلية بنسبة 19 F MR الطيفي .

ثمانية عشر إلى واحد وعشرين ساعة بعد تطبيق أدمي، 19 الخلايا الجذعية F-المسمى (DC) تهاجر إلى العقدة الليمفاوية المأبضية تجفيف. حركة DC عبر القنوات اللمفاوية في العقدة الليمفاوية politeal استنزاف يمكن أن يكون موضع تقدير من قبل تتراكب 1 H الصور التشريحية مع 19 F ا?...

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Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

وقد اكتسب تتبع من الخلايا باستخدام التصوير بالرنين المغناطيسي اهتمام ملحوظا في السنوات الماضية. يصف هذا البروتوكول بوضع العلامات على الخلايا الجذعية مع الفلور (<sup&gt; 19</sup&gt; جزيئات F) الغنية، وتطبيق في الجسم الحي من هذه الخلايا، ورصد مدى هجرتهم إلى العقدة الليمفاوية استنزاف مع<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H التصوير بالرنين المغناطيسي و<sup&gt; 19</sup&gt; F MRS.

رصد هجرة الخلايا الجذعية باستخدام<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H التصوير بالرنين المغناطيسي

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

monitoring-dendritic-cell-migration-using-19f-1h-magnetic-resonance

Research

JoVE Journal

Immunology and Infection

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

11.7K Views.  A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine. وقد اكتسب تتبع من الخلايا باستخدام التصوير بالرنين المغناطيسي اهتمام ملحوظا في السنوات الماضية. يصف هذا البروتوكول بوضع العلامات على الخلايا الجذعية مع الفلور ( 19 جزيئات F) الغنية، وتطبيق في الجسم الحي من هذه الخلايا، ورصد مدى هجرتهم إلى العقدة الليمفا...

Video: Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

غير الغازية تصوير الزاجل كريات بيضاء والهجرة في الجسم الحي</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

في الوقت الحقيقي التصوير باء Leukotriene 4 الهجرة خلية وساطة BLT1 التفاعلات مع arrestin - β

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

الزاجل فحوصات تنافسية لدراسة غوت - مدار هجرة الخلايا التائية

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

بروتوكول الأمثل لترنسفكأيشن كفاءة الخلايا الجذعية من دون نضوج الخلايا

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

تحضير العينة من<em&gt; المتفطرة السلية</em&gt; خلاصات لدراسات النووية المغناطيسي الرنين Metabolomic

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

تحليل الرئوي نضوج خلية شجيري والهجرة خلال التهاب الشعب الهوائية التحسسي

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

جيل من لقاح جديد للخلايا التشجرية باستخدام الميلانوما والخلايا الجذعية السرطانية الحرشفية

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

التصوير CD4 T خلية الخلالية الهجرة في الأدمة الملتهبة

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

مقايسة على أساس CFSE لدراسة الهجرة من الجلد الفئران الخلايا الجذعية في تصريف الغدد الليمفاوية خلال العدوى مع<em&gt; المتفطرة البقرية</em&gt; عصيات كالميت غيران

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

توليد عدد كبير من فروعه النقوي وسلائف الخلايا الجذعية من نخاع العظام مورين باستخدام خلية رواية فرز استراتيجية

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...