Flotation-Based T Cell Isolation Using Buoyancy-Activated Cell Sorting

Abstract

Source: Snow, T., et al. Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles. J. Vis. Exp. (2022)

This video demonstrates T cell isolation from peripheral blood mononuclear cells (PBMCs) using the functionalized buoyancy-activated cell sorting (BACS) technology. The isolation technique involves the positive selection of CD3+ T cells by labeling the cells with biotinylated anti-CD3 antibodies and separating the antibody-bound cells using streptavidin-bound microbubbles.

Protocol

1. Isolation of T cells with microbubbles using positive selection

NOTE: This protocol details a small-scale CD3⁺ positive selection approach using SAMBs.

1. Incubate 3 x 10⁸ commercially obtained PBMCs in 2.5 mL of separation buffer with biotinylated anti-CD3 (OKT3) antibody at a concentration of 25 ng of antibody per 1 million cells (25 ng/M). Gently mix by pipetting up and down, and incubate at room temperature for 10 min.
2. Add streptavidin microbubbles (SAMBs) at a ratio of 0.5 (SAMB quantity):1 (cell quantity) according to the manufacturer's reported SAMB concent....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biotin anti-human CD3 (OKT3) Antibody	Biolegend	317320
Leukopak	BioIVT	HUMANLMX100-0001129
Normal Human PBMCs	BioIVT	HUMANHLPB-0002562
Streptavidin Microbubble Kit (includes Akadeum's separation buffer)	Akadeum	11110-000