Isolation and Purification of B Cells from Human Peripheral Blood

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 demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

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. Isolation and Purification of Human Peripheral Blood B Cells

1. Draw ~ 10 mL of blood from the median cubital vein (in the cubital fossa anterior to the elbow) into a 15-mL tube containing K₂EDTA (1.5 to 2.0 mg/mL blood) and immediately invert the tube several times to prevent clot formation.
2. Add 35 mL of autoclaved (121 °C, 15 min) red blood cell (RBC) lysis buffer (1....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD Vacutainer K2E	BD Biosciences	367525	10 ml tube
Ficoll-Paque Plus	GE Healthcare	17-1440-02	Endotoxin-free
Trypan blue 0.5% solution	Biological Industries	03-102-1B	Dye
IMag Human B lymphocyte enrichment set	BD Biosciences	558007	Reagent
Streptavidin magnetic microbeads	BD Biosciences	9000810	Reagent
15 ml Falcon tubes	BD Falcon	352196	Plastic ware
Biotinylated CD27 mAb	Biolegend	302804	Clone O323
Biotinylated anti-human CD27	Biolegend	302804	Clone O323
Biotinylated anti-human CD27	eBioscience	13-0279-80	Clone O323