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Flow Cytometry Applications - Immunology

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Immunology

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Tissue-Specific Immune Environments

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The immune system, which mediates the body’s response to the introduction of foreign material, is made up of multiple cell types collectively called lymphocytes. Lymphocyte subtypes include B cells (which secrete antibodies), cytotoxic T cells, helper T cells (which secrete cytokines), and natural killer (NK) cells. Characterization of lymphocyte subtypes and cytokine signaling is essential for understanding the complex nature of the immune system.

Guava easycyte™ systems offer rapid interrogation of a variety of common immunologic assays, with up to 8 parameters of detection. Amnis imaging flow cytometers can readily analyze more complex assays, with up to 5-12 parameters, including visual verification of specific cells in suspension and rare events.

Multiparametric phenotypic analysis by flow cytometry allows researchers to study the dynamics of immune signaling in intact cells, and offers many unique capabilities, such as:

  • distinguish one subpopulation of cells within a heterogeneous mixture
  • evaluate impact of activation by antigens
  • detect suppression of normal immune activation or impact of disease state on phenotypes
3-Part Differential Capabilities

Dual platform approach to immunology. 3-Part Differential with antibody confirmation (lymphocytes red CD3, monocytes yellow CD14, granulocytes green CD15). Adult human blood stained with CD3PECY5, CD14-PE, and CD15-FITC lysed with the guava® lysing solution and acquired on the guava easyCyte™ instrument. Data displayed shows clear identification of the 3 major white blood cell populations.

Dual platform approach to immunology:
Data using the guava easyCyte™ instrument is complementary to Milliplex™ cytokine panels on the Magpix® system.


Download our newsletter containing an artlcle on immunology HERE.
Accuracy of CD4 count and CD4% values using the Auto CD4/CD4% assay.
Regression plots of CD4% data from guava® Auto CD4/CD4% test versus data from a comparative device, representing abnormal adult (top plot) and abnormal pediatric (bottom plot) samples.

AutoCD4/CD4% (Human CD4 T cell enumeration and percent)

Guava® AutoCD4/CD4% is an application for enumeration of CD4+ Human T cells in whole blood samples. This application is offered on a dedicated use instrument platform. It provides counts of CD4+ T Lymphocytes in whole blood as well as determination of the number of CD4+ T-cells as a percent of Total Lymphocytes.

Download our data sheet on AutoCD4 HERE.

Levels of apoptosis, assessed by Annexin V response, in CD4 and CD8 subsets for multiple compounds are depicted in heat map.
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Immunotoxicology

T-cell mediated cytotoxicity plays a critical role in down regulation of the immune response. Moreover, immunosuppressant compounds often mediate their effects through immunotoxicity. We have developed a broad range of kits for evaluation of immunotoxicology through a variety of mechanisms.

  • Interested in Kits for Immunotoxicology? Click here.

In the example to the right, PBMCs were treated with 80 different cytotoxic compounds for 20 hours. Cells were analyzed with the FlowCellect™ Human T Cell Apoptosis and CD4 T Cell FAS assays followed by microcapillary cytometry. Levels of apoptosis, assessed by Annexin V response, in CD4 and CD8 subsets for multiple compounds are depicted in heat map. The table summarizes compounds that demonstrate significant apoptosis and the % of CD4 and CD8 T cells that underwent apoptosis.

Download our poster on immunotoxicology HERE.

Human B Cell Function

Memory B cells represent approximately 30-60% of the B-cell pool in healthy donors. B-cell subpopulations are reported to be defective in patients suffering from immuno-deficiency disorders, which are correlated with reductions in numbers of circulating CD19+CD27+ memory B cells. For evaluation of Human B cells, we now offer a variety of options.

Identification of Human Memory B cells by using specific phenotypic CD markers

Isolation and Identification of Human Memory B cells from human PBMSs.

Isolation and Identification of Human Memory B cells from human PBMSs. Human memory B cells are phenotypically identified by using specific human CD markers: Human cd5, Cd19, and Cd27. CD 27 has been identified as the key marker for identifying memory B cells. In (A), lymphocyte populations are gated, and B cells from a normal patient sample are shown using bivariate analysis plotting CF5+cd19+(i). Memory B cells subsets from a normal patient are also identified by using a CD+CD27+ (ii). In healthy patients, memory B cells represent approximately 30-60% of the B-cell pool. B-cell subpopulations are defective in patients suffering from immunodeficiency disorders, showing a reduced number of circulating CD19+cd27+ memory B cells.

Download our poster on human B cell function HERE.

The histograms illustrate how using EMD Millipore's FlowCellect™ GPCR Surface Indentification Kit can be used to quantitatively measure cell surface expression for any given cell type.
The histograms to the right illustrate how using Merck's FlowCellect™ GPCR Surface Indentification Kit can be used to quantitatively measure cell surface expression for any given cell type. The histogram on the far right (pink) represents the positive control cells, the histogram in the middle (green) represents the native cell line (Jurkat T cells), and the histogram on the far left (red) is the negative control cells.

Quantitation of Chemokine Receptor Expression

GPCR surface identification flow cytometry kits provide high quality, reproducible data in far less time than traditional methods for chemokine receptor quantitation, while avoiding the hazard and expense of radioligand binding assays. Now you can identify and quantify GPCRs on the surface of any cells using a GPCR-specific antibody validated for flow cytometry, and use the included positive and negative controls cells. Merck offers FlowCellect™ Kits for quantitation of Chemokine Receptor Surface Expression.

Download our poster on quantitation of chemokine receptor expression HERE.