L&#39;autore desidera ringraziare la signorina Lora Matthews per eseguire la tecnica e il signor Paolo Pastore per la cinematografia. Questo progetto è stato finanziato dalla University of South Australia, il programma di Medicina di Laboratorio.

Preparazione di 5x Veronal Buffered Saline (VBS) <ol><li> Per preparare il veronal Buffered Saline (VBS), tre soluzioni separate devono essere preparati. </li><li> 1 Preparare la soluzione sciogliendo 21.25gm di NaCl e 0.94gm Barbitone di sodio in 350 ml di acqua distillata. Le concentrazioni finali dei Barbitone NaCl e sodio sono 1,02 e 13 mm rispettivamente. </li><li> Preparare la soluzione 2 sciogliendo 1.44gm di Barbitone in 125ml di acqua distillata calda. La concentrazione finale di Barbitone è 62,5 mm. </li><li> 3 Preparare la soluzione sciogliendo 20.33gm di MgCl 2 e 4.41gm di CaCl 2 in 100 ml di acqua distillata. La concentrazione finale di MgCl 2 e CaCl 2 è 2.18m e 440mm rispettivamente. </li><li> Mix di soluzioni 1 e 2 e raffreddare a temperatura ambiente. </li><li> Una volta che la soluzione combinata si è raffreddato, aggiungere 1,25 ml di soluzione al 3 e regolare il pH a 7,3-7,5 usando HCl 1M. </li><li> Regolare il volume finale di 500ml con acqua distillata per preparare una soluzione 5x magazzino. </li><li> Per preparare una soluzione 1x di lavoro, diluire il magazzino 1:05 con acqua distillata. </li></ol> Diluizione del Revercells <ol><li> Risospendere delicatamente la soluzione stock di Revercells per inversione e quindi rimuovere 1ml di cellule e centrifugare per 5 min a 600 g a 4 ° C. </li><li> Dopo la centrifugazione, rimuovere con attenzione il sopranatante e risospendere le cellule in 3 ml di 1x veronal Buffered Saline. </li><li> Le celle possono essere conservati a 4 ° C fino all&#39;utilizzo. </li></ol> Procedura <ol><li> Trasferire 50ml di 1x VBS in righe A1-A12 di una piastra di fondo ben 96 U. </li><li> Per Bene A1, aggiungere 50μl di siero anti-A e mescolare accuratamente con una pipetta. </li><li> Utilizzando un nuovo puntale per pipetta, rimuovere 50μl di liquido dal pozzetto A1 e trasferirlo a ben A2. Agitare bene e ripetere questo processo fino a raggiungere ben A11 e poi gettare il 50μl ultimi da questo bene. </li><li> Il campione di siero è stato diluito in serie di 2 volte da 1:2 a 1:2048. Nessun siero viene aggiunto al Bene A12 in quanto questo è il controllo VBS negativo. </li><li> Se più di un campione di siero deve essere valutata, ripetere i passaggi 4,1-4,4 per riga B, ecc </li><li> Una volta che tutte le diluizioni sono state fatte, aggiungere 50μl di 1% Revercells a tutti i pozzetti. Nota: Il Revercells stabilirà il tempo e devono essere delicatamente sospesi prima di essere distribuiti nei pozzetti. </li><li> Miscelare i campioni picchiettando delicatamente sul lato del vassoio microtitolazione, coprire e lasciare la reazione di emoagglutinazione a procedere per 1h a 37 ° C. Una sala incubatore o opportunamente riscaldata può essere utilizzato. </li><li> Dopo l&#39;incubazione, rimuovere con attenzione il vassoio dal termostato ed esaminare la piastra per emoagglutinante. Il controllo VBS bene dovrebbe essere il primo bene esaminato. Se la reazione è negativa, allora i risultati sono validi. Se il campione VBS evidenzia un risultato positivo emoagglutinazione, allora i risultati non possono essere utilizzati ed il processo deve essere ripetuto. Una reazione positiva emoagglutinazione apparirà come un largo foglio sul fondo del pozzo, mentre una reazione negativa apparirà come una piccola pallina di cellule concentrate nel centro del pozzo. Un risultato intermedio avrà un alone di cellule positive con un nucleo centrale di cellule pellet. Questo risultato si verifica come il siero ha qualche Ab che possono reagire, ma quantità insufficiente di Ab per provocare una reazione completa. </li><li> La sezione rappresentante risultati ha due rappresentazioni diagrammatiche dei risultati attesi. Un esempio di un insieme valido e valido dei risultati è fornito. Il titolo per l&#39;anti-siero saranno anche determinati. </li></ol> Rappresentante dei risultati: Il video contiene un esempio di risultati rappresentativi. Qui di seguito (Fig. 1 e Fig. 2) sono due esempi mostrati in maniera schematica. Il primo esempio è di una reazione che non può essere interpretato come il controllo VBS è positivo, mentre il secondo è un test valido che permette il titolo da definire. <img src="/files/ftp_upload/1752/1752fig1.jpg" alt="Figura 1" /> Figura 1. Un esempio di emoagglutinazione in cui il risultato positivo non è riuscito. Nella rappresentazione schematica sopra, la reazione heamagglutination non può essere accolto. Il controllo VBS siero negativo mostra una reazione positiva emoagglutinazione come dimostra la formazione del foglio alla base del pozzo. Ciò indica che un errore si è verificato ed i risultati non possono essere accettati. In questo caso, il processo deve essere ripetuto. <img src="/files/ftp_upload/1752/1752fig2.jpg" alt="Figura 2" /> Figura 2. Un esempio di emoagglutinazione in cui il risultato positivo ha funzionato correttamente. Nella rappresentazione schematica, la reazione heamagglutination è valido. Il controllo VBS negativo mostra una reazione negativa emoagglutinazione unos dimostrano i pellet che si è formata alla base del pozzo. Il pozzo è positivo ultima riga 7, che indica che il titolo di questo campione di siero è 128 (ovvero il reciproco del 1 / 128). Questo sarebbe un esempio di un anticorpo che è presente in quantità moderate nel siero.

Il saggio di emoagglutinazione è una tecnica molto semplice che permette un gran numero di campioni di siero da valutare in un breve lasso di tempo. Ha anche il vantaggio di essere letto senza la necessità di attrezzature specializzate, con l&#39;occhio idoneo. Questa tecnica può anche essere modificato per il rilevamento di ormone umano della gonadotropina corionica presenti nelle urine, come parte di una prova di inibizione dell&#39;emoagglutinazione. In relazione ad un test di emoagglutinazione standard, ci sono a...

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<td>Revercells</td>
<td>&nbsp;</td>
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<td>Use at 1% final</td>
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<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Use at 1x final</td>
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determining the reactivity and titre of serum using a haemagglutination assay

Emoagglutinazione è una forma specifica di agglutinazione ed è utilizzato quando gli anticorpi si legano ai globuli rossi, che agiscono come un antigene particolato. I globuli rossi sono bersagli particolarmente utili in quanto sono facilmente disponibili e agglutinazione è osservabile ad occhio nudo. Questa tecnica viene comunemente utilizzata per determinare la concentrazione di un anticorpo (Ab), per gruppo sanguigno e la quantificazione virale. In questo video, le fasi coinvolte nella preparazione e nella esecuzione di un test di emoagglutinazione è dimostrata utilizzando anticorpi specifici per gruppo sanguigno A-antigeni aggiunto ai globuli rossi (Revercells). L&#39;antisiero è diluito in una serie ben 96 U-basso vassoio microtitre, a cui si aggiunge una sospensione di Revercells. I campioni sono miscelati e poi incubate a 37 ° C per 60 minuti. Trascorso questo tempo, i campioni possono quindi essere facilmente segnato per ve, ve + e intermedi reazioni emoagglutinazione (-/+). Questo approccio permette la reattività e titolo di un campione di siero deve essere valutato con una tecnica rapida e semplice. Il video coprirà la teoria alla base del test, come i risultati vengono letti e interpretati, come determinare il titolo, come il dosaggio può essere modificato ed eventuali problemi associati con l&#39;uso di questa tecnica.

Watch this Scientific Journal Video about Determining the Reactivity and Titre of Serum using a Haemagglutination Assay at JoVE.com

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Emoagglutinazione è una forma di agglutinazione in cui gli anticorpi si legano ai globuli rossi. Globuli rossi sono sempre disponibili ed i risultati sono facilmente osservabili ad occhio nudi. Questo video dimostra i passi coinvolti in un test di emoagglutinazione, interpretando i risultati e la determinazione del titolo.

Determinazione della reattività e Titolo di siero usando un test dell&#39;emoagglutinazione

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

determining-reactivity-titre-serum-using-haemagglutination

Research

JoVE Journal

Immunology and Infection

20.9K Views.  University of South Australia. Emoagglutinazione è una forma di agglutinazione in cui gli anticorpi si legano ai globuli rossi. Globuli rossi sono sempre disponibili ed i risultati sono facilmente osservabili ad occhio nudi. Questo video dimostra i passi coinvolti in un test di emoagglutinazione, interpretando i risultati e la determinazione del titolo.

Video: Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

Misura del Complemento 50% emolitica (CH 50) Attività di siero

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

Ricerca

Immunologia e infezioni

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Determinazione dell&#39;attività fagocitaria dei Campioni anticorpi clinici

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Determinazione ottimale attività citotossica di specifiche cellule T umane Her2neu CD8 confrontando il Cr51 saggio di rilascio al Sistema xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Induzione di Graft-versus-host disease e<em&gt; In Vivo</em&gt; T monitoraggio cellulare Utilizzando un MHC-abbinato modello murino

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un<em&gt; In Vitro</em&gt; Aderenza Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Pairwise crescita Concorso test per la determinazione della Fitness replica del virus dell&#39;immunodeficienza umana

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

High-throughput Quantitative Real-time RT-PCR test per la determinazione di espressione Profili di tipo I e III interferone sottotipi

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

La valutazione della retina microglia fagocitaria Funzione<em&gt; In Vivo</em&gt; Usando un test citometria a flusso basato su

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Ottimizzazione di un Quantitative Micro-neutralizzazione Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Determinazione della reattività e Titolo di siero usando un test dell'emoagglutinazione

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Capitoli in questo video

Misura del Complemento 50% emolitica (CH₅₀) Attività di siero

Determinazione dell'attività fagocitaria dei Campioni anticorpi clinici

Determinazione ottimale attività citotossica di specifiche cellule T umane Her2neu CD8 confrontando il Cr51 saggio di rilascio al Sistema xCELLigence

Induzione di Graft-versus-host disease e

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un

Pairwise crescita Concorso test per la determinazione della Fitness replica del virus dell'immunodeficienza umana

High-throughput Quantitative Real-time RT-PCR test per la determinazione di espressione Profili di tipo I e III interferone sottotipi

La valutazione della retina microglia fagocitaria Funzione

Ottimizzazione di un Quantitative Micro-neutralizzazione Assay

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione