Flow Cytometry-Based Cell Sorting for Different Circulatory B Cell Populations

Protokol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Cell Sorting for the Collection of Naïve B Cells, Memory B Cells, and Plasmablasts/Plasma Cells (PBs/PCs)

1. Using purified human peripheral blood B cells, determine the cell number using a hemocytometer or an automatic cell counter.
2. Resuspend the cells in cold PBS buffer at the concentration of 10⁷ per mL in a 5-mL polystyrene tube.
3. Add 1 - 2 µg of human IgG per 10⁶ cells and incubate on ice for 10 min for the Fc block.
4. Add 1 µg each of anti-CD19-APC (clone: HIB19), anti-CD27-eFluor450 (clone: O323), and anti-CD38-PE (clone: HIT2) per 10⁶ cells; mix well and incubate on ice for 30 min.
5. In the last 5 min in step 1.4, add 5 µL of the commercial 7-amino actinomycin D (7-AAD).
6. Add 2 mL of PBS to the tube, vortex, and centrifuge at 600 x g for 5 min.
7. Resuspend the cells in sorting buffer (sterile PBS with 2% BSA and 2 mM EDTA) at a concentration of 1 - 5 x 10⁷ cells per mL in a 15-mL tube.
8. Filter the cells through a nylon mesh cell strainer (40 µm pore size) to eliminate cell clumps.
9. Separate the cells with a flow cytometric sorter equipped with three lasers: violet (405 nm), blue (488 nm), and red (640 nm).
   NOTE: The blue laser alone is sufficient for 3-color flow cytometry.
10. Sort the cells into three 15-mL tubes (containing 5 mL of RPMI medium) for the simultaneous collection of naïve B cells (CD19⁺CD27^-), memory B cells (CD19⁺CD27⁺), and PBs/PCs (CD19⁺CD27^+/hiCD38⁺).
    NOTE: Sorted naïve and memory B cells can be further cultured.

Malzemeler

Name	Company	Catalog Number	Comments
15 mL Falcon tubes	BD Falcon	352196
Blue nylon mesh cell strainer, 40 μm	BD Falcon	352340
Anti-human CD19-APC	Biolegend	302212	clone HIB19
Anti-human CD27-eFluor 450	eBioscience	48-0279-42	clone O323
Anti-human CD38-PE-Cy7	Biolegend	303516	clone HIT2
Anti-human CD38-PE-Cy7	BD Biosciences	560677	clone HIT2
Anti-human CD45-FITC	Biolegend	304006	clone HI30
Anti-human CD45-FITC	BD Biosciences	555482	clone HI30
Anti-mouse/rat/human CD27- PerCP Cy5.5	Biolegend	124213	clone LG.3A10
Anti-human CD27-PerCP Cy5.5	BD Biosciences	65429	clone L128
Anti-human CD19-FITC	Miltenyi Biotec	130-098-064	clone LT19
Anti-human CD19-FITC	GeneTex	GTX75599	clone LT19
Anti-human CD20-FITC	BD Biosciences	555622	clone 2H7
7-aminoactinomycin D (7-AAD)	BD Biosciences	559925
Human IgG	Jackson ImmunoResearch	009-000-003