Encyclopedia of Experiments: Immunology
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Transcript
To perform immunomagnetic negative separation of B cells, collect human peripheral blood in an anticoagulant-containing tube to prevent blood clotting. Add red blood cell, RBC, lysis buffer to lyse the RBCs without affecting other blood cells.
Centrifuge to sediment the intact, denser blood cells from the less dense lysed RBCs, which remain in the supernatant. Discard the supernatant. Resuspend the pellet in fresh medium.
Transfer the cells to a culture plate. Incubate to allow the macrophages and monocytes to adhere to the plate while the non-adherent cells, including B cells, remain in suspension.
Collect the cells in suspension into a fresh tube. Centrifuge to pellet the cells and resuspend them in a buffer.
Add a cocktail solution containing biotinylated antibodies specific for blood cells other than B cells. The antibodies bind to antigens on the surface of non-B cells, leaving the B cells unlabeled.
Centrifuge. Discard the supernatant containing unbound antibodies.
Add a solution of streptavidin-conjugated magnetic beads. During incubation, the streptavidin on the microbeads binds tightly to the biotinylated antibodies bound to non-B cells, forming complexes.
Place the tube in a magnetic field to adhere the magnetic beads-bound non-B cells to the tube walls, leaving the B cells in suspension. Transfer the supernatant containing pure B cells to a tube.
Centrifuge and resuspend the B cells in medium for further downstream analysis.
