Method Article

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

July 8th, 2025

In This Article

Abstract

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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

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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 hum....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
15 mL Falcon tubesBD Falcon352196
Blue nylon mesh cell strainer, 40 μmBD Falcon352340
Anti-human CD19-APCBiolegend302212clone HIB19
Anti-human CD27-eFluor 450eBioscience48-0279-42clone O323
Anti-human CD38-PE-Cy7Biolegend303516clone HIT2
Anti-human CD38-PE-Cy7BD Biosciences560677clone HIT2
Anti-human CD45-FITCBiolegend304006clone HI30
Anti-human CD45-FITCBD Biosciences555482clone HI30
Anti-mouse/rat/human CD27- PerCP Cy5.5Biolegend124213clone LG.3A10
Anti-human CD27-PerCP Cy5.5BD Biosciences65429clone L128
Anti-human CD19-FITCMiltenyi Biotec130-098-064clone LT19
Anti-human CD19-FITCGeneTexGTX75599clone LT19
Anti-human CD20-FITCBD Biosciences555622clone 2H7
7-aminoactinomycin D (7-AAD)BD Biosciences559925
Human IgGJackson ImmunoResearch009-000-003

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Tags

Flow CytometryCell SortingB Cell PopulationsNaive B CellsMemory B CellsPlasmablast Plasma CellsCD19 CD27 CD38Fluorescent AntibodiesDead Cell MarkerFACS Buffer

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