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.