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.
- 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.
- Add streptavidin microbubbles (SAMBs) at a ratio of 0.5 (SAMB quantity):1 (cell quantity) according to the manufacturer's reported SAMB concentration.
- 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.
- 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.
Disclosures
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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Cite This Article
Flotation-Based T Cell Isolation Using Buoyancy-Activated Cell Sorting. J. Vis. Exp. (Pending Publication), e21528, doi: (2023).