How to Characterize Immune Responses With Flow Cytometry

How to use flow cytometry to characterize the immune response Including experimental design, antibody selection and gating of subgroups If you are facing challenges, you need to use flow cytometry Design antibody detection kits to analyze intracellular signal transduction And you are not sure what to do We can help you Let's take a look at an experimental example Show you how to use multiple target flow cytometry Construct an antibody detection kit to assess immune cell activation You can also visit the website cst-science.com/youcanflow Download supporting application note Multiple target determination can enhance the effect of flow cytometry analysis Phenotypic surface markers can pinpoint which cell populations Respond to processing or experimental interference Simultaneous intracellular reading Can also detect signal transduction pathways related to this response In our example our goal is Assess T lymphocyte activation in heterogeneous PBMC samples You can choose the appropriate surface markers to start building a test set This allows you to gate cell populations and subpopulations in the analysis In our example we use CD3 as the total T cell marker Using CD4 as a helper T cell marker Add one or more intracellular targets Activated as a signal transduction pathway Reading of protein expression or other biological functions Phospho-specific antibodies To detect phosphorylation of SLP76 as a readout of T cell activation Finally consider in your antibody testing portfolio Add a dye to identify live/dead cells such as Ghost Dye So that dead cells can be excluded from the analysis Here are some Best practices to ensure your flow cytometry results are reliable and repeatable Make sure to choose when matching your antibody test kit Antibodies or antibody conjugates that have been validated by flow cytometry Performance in other applications cannot be represented The performance of the antibody in flow cytometry Check your antibody test kit Are all antibodies suitable for your experiment Fixed and transparent processing conditions used If there is no such information May need to test and optimize experimental procedures Finally, when choosing fluorescein Avoid spectral overlap as much as possible Or in your analysis if necessary Add fluorescence compensation Prepare live PDMC cells for flow cytometry analysis We first incubate the cells with Ghost Dye and wash Then we processed it with cross-linked CD3/CD28 Activate T cells Fixed after 15 minutes Permeabilization and antibody labeling To analyze SLP76 activity in T cells We apply sequential gate design Use forward/inward scattering in gate 1 to select lymphocytes Use Ghost Dye to select live/dead cells in gate 2 Then select for CD3 expression in gate 3 Select for CD4 expression in gate 4 Gating in this way allows you to quickly focus on relevant cell populations And get important data T cell activation in our experiment Clearly identifiable in CD3/CD4 positive T cells And after activation in the gating cell population Positive expression of phosphorylated SLP76 Cell proportion is higher than normal PBMC For a more detailed guide to design your flow cytometry test suite Please visit cst-science.com/youcanflow to download the application note

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