Encyclopedia of Experiments: Immunology
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Transcript
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.
