This article describes a method for isolating T cells from peripheral blood mononuclear cells (PBMCs) using buoyancy-activated cell sorting (BACS). The process involves the use of biotinylated antibodies and functionalized microbubbles to selectively capture T cells while minimizing stress on the cells.
To isolate T cells — white blood cells essential to the immune system — using buoyancy-activated cell sorting, BACS, take a suspension of peripheral blood mononuclear cells, PBMCs. PBMCs contain target T cells and non-target cells like B cells, monocytes, natural killer cells, and dendritic cells.
Add a buffer containing an adequate amount of biotinylated anti-CD3 antibody and incubate for the required duration. The antibody binds to the CD3 complex — a multimeric protein complex on the cell surface — a defining feature of T cells.
Add functionalized microbubbles — small spherical particles coated with streptavidin — for positive selection of antibody-bound T cells. The core of each microbubble is a hollow sphere, making them low-density particles capable of floating in an aqueous solution.
Incubate the mixture under agitation, allowing the microbubbles and sample to mix thoroughly. During incubation, streptavidin on the microbubble binds to biotin on the T cell-bound antibody, immobilizing the cells on the microbubbles.
Centrifuge the tube. Non-target cells separate from the mixture as a pellet at the bottom.
The target T cell-bound microbubbles stay afloat on the surface, leading to buoyancy-based separation of the T cells. This separation technique limits stress on the target cells. With a thin pipette, remove the pellet and subnatant without disturbing the microbubble layer.
Resuspend the isolated, microbubble-bound T cells in an appropriate culture medium for further processing.
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.
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