The appropriate controls for this multicolor flow cytometry experiment include an unstained sample to look at autofluorescence, single stained samples to allow compensation data to be generated, isotype controls to check for non-specific background staining and FMO controls to account for spreading of the data. A list of the antibodies used can be found in Table 1. To identify peripheral blood B cell subsets, markers were first identified from the literature which would allow the identification of specific B cell subsets including naïve, memory, class switched and non-class switched, transitional B cells and plasmablasts. This includes careful sample prep, knowing the biology of your sample, understanding your flow cytometer configuration and performing the right controls. Successful flow cytometry panel building relies on careful planning prior to staining. For more information on B cell lineage markers, function and activation, including all our antibodies useful for identification of B cells please go to our dedicated B cell page. Additional markers allow identification of secondary lymphoid organ B cell populations, for instance CD24 and CD21, for marginal zone B cells, and CD69, CD80 and CD86 for activation status.
With the addition of more markers, such as CD27, CD24, CD38 and surface immunoglobulin, specific blood subsets such as naïve, memory and transitional B cells can be identified. However the key marker for identification of human B cells is CD19, which is expressed by virtually all B cell lineages (in mouse the key pan B cell marker is CD45R, also known as B220, and in rat it is CD45RA). The first marker to be identified, using monoclonal antibodies specific for B cells, was B1, now known as CD20. This guide to immunophenotyping of human B cells takes you through some of the common markers and gating strategies used to identify B cells by flow cytometry, with examples of data acquired on the ZE5™ Cell Analyzer. Click here for an overview of B cells from discovery to therapyī cell subsets can be difficult to identify due to variability and low level expression of markers and the rarity of some populations.
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