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

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.

Materials

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