Common marking and gating strategies
Together with T lymphocytes, B lymphocytes form the adaptive immune system. They are heterogeneous cell populations with unique functional properties. Their main function is to produce antibodies, but they can also be professional antigen-presenting cells of T-lymphocytes that help induce an effective immune response. Abnormalities in B cell development or function contribute to a variety of autoimmune and malignant diseases, and their identification is critical for disease treatment and detection of residual disease.
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B cell subsets can be difficult to identify due to marker variability and low expression and rarity of certain populations. This guide to human B cell immunophenotyping will walk you through some common markers and gating strategies used to identify B cells using flow cytometry, with examples of data acquired using the ZE5™ cytometer.
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The first marker identified using a B-cell specific monoclonal antibody was B1, now known as CD20. However, the key marker for human B cell recognition is CD19, which is expressed by almost all B cell lineages (in mice the B cell marker is CD45R, also known as B220, and in rats it is CD45RA). By adding multiple markers such as CD27, CD24, CD38 and surface immunoglobulin, specific blood subpopulations such as naïve B cells, memory B cells and transitional B cells can be identified. Additional markers identify secondary lymphoid organ B cell populations such as CD24 and CD21 for marginal zone B cells and CD69, CD80 and CD86 in the activated state. For more information on B-cell lineage markers, function and activation, including all of our available antibodies for B-cell recognition, visit ourDedicated B cell site。
Successful setup of a flow cytometry panel requires careful planning prior to staining. This includes careful sample preparation, understanding the biology of the sample, understanding the configuration of the flow cytometer, and running appropriate controls.
To identify B cell subsets in peripheral blood, markers from the literature were first identified that would allow for the identification of specific B cell subsets, including naïve, memory, class-switching and non-class-switching, transitional B cells, and plasma mother cell. The list of antibodies used is given in Table 1.
Appropriate controls for this multicolor flow cytometry experiment include unstained samples to visualize autofluorescence, single stained samples to generate compensation data, isotype controls to control non-specific background staining, and FMO to account for data scatter control. In addition, human serum was used to block Fc receptors on B cells and a viability dye was used to remove dead cells. The FMO control of CD24PE is shown in Figure 1 below.
As shown in the picture. 1. CD24 PE FMO control.Cells were stained with all antibodies in the group except CD24 PE (MCA1379PE) were replaced by isotype controls. This allowed us to confirm that CD24 positive cells were not due to fluorescence scatter in the final staining.
Device configuration can affect the availability of fluorophores used in multicolor panels. TheCell analyzer ZE5With 5 lasers and 27 fluorescence parameters, it allows the spread of the fluorophores used across the lasers and filters to minimize fluorescence wastage. In addition, a spectral viewer included in the software allows the user to visualize possible conflicts between fluorophores, see Figure 2. In addition, common markers such as CD19 are placed on relatively faint fluorophores to allow better resolution of less common markers with bright fluorophores.
Figure 2. ZE5 cell analyzer spectrometer. Emission curve for a fluorophore selected in the B Cells panel to display the fluorophorepolluteand cross laser excitation and emission.
To further increase the resolution, we added the dump channel. These include CD3, CD14, and CD56, all stained with an A700-conjugated antibody. This allowed cells positive for these markers to be excluded from the analysis. Cells to be stained should be as fresh as possible and stored under conditions that ensure cell health. Optimize the staining protocol by titrating all antibodies to the point that yields the optimal staining index, which is the difference between the mean fluorescence intensities of the positive and negative populations.
The frequency of B cells in human peripheral blood is 3-10%, with some subpopulations (e.g. memory B cells) representing only a small fraction of this population. Hence we get 1.2 x 106cells to ensure we can visualize and accurately capture all relevant populations.
A list of selected markers, fluorophores and antibody titration data is provided in Table 1 below.
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Reinigung, A488, A647, A700, APC, FITC, PB, PE
Cleaning, FITC, PE
Cleaned, A488, A647, FITC, PE
Rein, A647, FITC, PB, PE, PE-A647, PE-A750
Cleaning, A488, A647, FITC
410/450, 408/512, 398/550, 498/521, 547/573, 583/603, 649/660
Abbreviations: Axxx, Alexa Fluor; APC, allophycocyanin; FITC, fluorescein isothiocyanate; PB, pacific blue; PE, phycoerythrin; Pur., cleaned; PerCP, polymethylchlorophyll.
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An initial basic gating strategy was used to identify B cells and exclude dead and duplicate cells collected during acquisition. In addition, T cells, NK cells and monocytes can be excluded using the dump channel as shown in Figure 3.
As shown in the picture. 3. Basic gating strategy. exist,Lymphocytes were identified by FSC and SSC.Secondto exclude dead cells.Cto exclude duplicates.DB cells were identified as positive for CD19 and negative for CD3, CD14 and CD56.
Specific subpopulations were identified by further sequential gating based on surface expression of different markers as shown in Figure 4, allowing identification of naïve and memory cells, class-switched and non-class-switched memory B cells, transitional B cells, etc. mother cell. The importance of obtaining large data sets can be seen from the extremely small number of plasmablasts, which represent less than 0.01% of the total number of cells obtained and only 0.1% of all B cells.
As shown in the picture. 4. Characterize B cells using an 8 color panel.Human peripheral blood stained with CD19 PB, CD20 PE-Cy5, CD24 PE, CD27 A647, IgD FITC, CD38 PE-A750, CD3, CD14 and CD56A700 and viability dye VivaFix 353/442 to identify different B cell subsets . exist,CD27 and IgD can recognize memory B cells and naïve B cells.Second,CD20CD38 recognizes plasmablasts.C, Representative overlays of naïve, memory, transitional and plasmablast populations and absolute numbers of various cell subsets. Data were collected with a ZE5 cell analyzer.
Using eight color panels, we were able to successfully identify multiple B cell subsets in human peripheral blood. For more information on human and mouse B cell subsets, lineages, and maturational markers, visit our websiteDedicated B cell site。
In addition to antibodies for B cell detection, we have over 4,000 flow cytometrically validated antibodies to help you identify the cells shown here and more. To learn more about our antibodies, antibody cocktails, kits and isotype controls visit our dedicated websiteflow cytometryPage.
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