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

Published: July 31, 2023

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 concentration.
  3. Mix using a commercial end-over-end (EOE) rotator at 20 rpm for 10-15 min at room temperature. Centrifuge for 5 min at 400 x g at room temperature.
  4. After centrifugation, the positively selected cells will be at the top of the suspension with the SAMBs. The remaining non-selected cells will be in the cell pellet at the bottom of the tube. Using a 9in glass pipette, insert the tip below the bubble-cell layer to the bottom of the tube, aspirate the cell pellet and subnatant with an electronic pipette, and transfer them to a new tube.

Offenlegungen

The authors have nothing to disclose.

Materials

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

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Diesen Artikel zitieren
Flotation-Based T Cell Isolation Using Buoyancy-Activated Cell Sorting. J. Vis. Exp. (Pending Publication), e21528, doi: (2023).

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