I want to evaluate B cell activation by flow cytometry using B cells in human peripheral blood.
I have difficulty in establishing a positive control.
What is the best stimulator? Lipopolysaccharides, B-cell Activating factor, or other reagents?
What kind of markers are necessary for evaluating activation by flow cytometry?
CD19, CD27, CD24, CD38, IgD and IgM are enough for it?
Hello, Hiroyuki!
You see in this chapter, please:
Article Analysis by Flow Cytometry of B-Cell Activation and Antibody...
B-Cell Labeling and Stimulation
A fl uorescent dye to label B cells to track their cell divisions, e.g., carboxyfl uorescein succinimidyl ester (CFSE; Life Technologies). 2. TLR ligands include: LPS from E. coli (e.g., serotype 055:B5, Sigma-Aldrich) used at 0.1–10 μg/ml; Pam 3CSK 4 (Invivogen) used at 0.1–1 μg/ml;
Flow Cytometry Acquisition and Analysis
Flow cytometer with at least 4 channels (e.g., BD FACSCalibur™), with 7 channels preferred (e.g., BD™ LSR II) and manufacturer’s data acquisition and analysis software. 2. Additional fl ow cytometry analysis software (e.g., FlowJo, Tree Star, Inc.). 3. Flow cytometry staining buffer: PBS with 1 % BSA. 4. Appropriate fl ow cytometry tubes or plates 5. A fl uorescent viability dye, such as 7-aminoactinomycin D (7AAD). 6. Antibodies to detect key B-cell markers: CD45R/B220 to mark B cells and reveal their developmental/differentiation stage(s); CD38 to mark germinal center B cells in vivo and germinal center-like B cells in vitro (where CD38 is downregulated); CD138 (syndecan-1) to mark plasmablasts and plasma cells; and IgM and other antibody isotypes relevant to the experiment. Other common markers used for various activation stages of murine B cells, such as CD19, PNA or GL7, can be used depending on experiment ( see Note 9). A 7-color staining configuration for the BD™ LSR II cytometer is: (a) CFSE (b) PE—anti-B200 (c) 7AAD (d) PerCP-Cy5.5—anti-IgM (or IgD) (e) PE-Cy7—anti-CD38 (f) APC—anti-IgG1 (or other IgG isotypes, IgE, IgA) (g) APC-Cy7—anti-CD138
Article A Nonsense N –Terminus NFKB2 Mutation Leading to Haploinsuff...
For B cell proliferation, cells were stimulated with anti-IgM (10 μg/ml, Jackson Lab; 109–006-129), CD40L (100 ng/ml, Enzo; ALX522–110-C010), CpG (0.5 μM, Enzo; ALX 746–006-C100), IL-4 (50 ng/ml, Peprotech; 200–04), and IL-21 (50 ng/ml, Peprotech; 200–21) as indicated in the figure. After incubation for 3–5 days (3 days for T cells and 4–5 days for B cells), cells were stained with fluorochrome-conjugated CD4, CD8, or CD19 (BD Biosciences, Clone RPA-T4, RPA-T8, HIB19 respectively) for 30 min at 4 °C (dark). Cells were washed with PBS twice, and cells were acquired and analyzed by flow cytometry (Becton Dickinson FACSCanto II) and FlowJo software (FlowJo 10.5.2, TreeStar).
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