Flotation-Based T Cell Isolation Using Buoyancy-Activated Cell Sorting
Transcript
To begin, incubate 3 x 108 commercially-obtained PBMCs in 2.5 milliliters of separation buffer with biotinylated anti-CD3 antibody. Gently mix the components by pipetting, and incubate them at room temperature for 10 minutes.
Add streptavidin microbubbles to the cells at a ratio of 0.5 to 1, according to the manufacturer's instructions, and mix using a commercial end-over-end rotator at 20 RPM for 10 to 15 minutes at room temperature. Centrifuge at 400 x g for 5 minutes at room temperature.
After centrifugation, the positively selected cells will be at the top of the suspension with the streptavidin microbubbles, and the remaining nonselected cells will be in the cell pellet at the bottom of the tube.
Using a 9-inch 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.
Resuspend the bubble cell layer left in the original tube in 1 milliliter of complete T-cell medium. Centrifuge the subnatant at 400 x g for 5 minutes at room temperature, and use it for indirect measurement of purity and recovery.
