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

Published: July 31, 2023

Abstract

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity. J. Vis. Exp. (2016).

In this video, we describe a protocol for the flow cytometry-based sorting of naïve B cell, memory B cell, and plasmablast plasma cell subpopulations from human peripheral blood B cells. The different B cell subpopulations can be distinguished based on fluorophore-based antibody binding to the CD19, CD27, and CD38 receptors expressed on the B cell surfaces, followed by cell sorting.

Protocol

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.

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Flow Cytometry-Based Cell Sorting for Different Circulatory B Cell Populations. J. Vis. Exp. (Pending Publication), e21524, doi: (2023).

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