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Flow cytometry Centrifugation and Washing

The centrifugation step pellets cells from the staining suspension, thus permitting removal of the supernatant fluid, and the washing of unbound antibody from the cells. The unbound antibody is removed by dilution in the sequential washing steps. Exact advice about centrifugation speeds is not possible, as different centrifuges take more or less time to reach full speed. In general, you want to spin cells down hard enough that the supernatant fluid can be removed with little loss of cells, but not so hard that the cells are difficult to resuspend. If staining in Eppendorf tubes, a high speed microfuge with an angled rotor can be used; 1-2 seconds is long enough to pellet most cells well. If the Eppendorf lids are lined up in the rotor so that the hinges all point outward, then the cell pellets (even if not visible) will all be at the hinge side of the tube after spinning. The supernatant fluid can then be removed with confidence by inserting a glass disposable ("Pasteur") pipette into the other (non-hinge) edge of the tube. The disposable pipette should be hooked up to suction such that the aspirated fluid will go into a trap containing bleach. After addition of 1 ml of S/W buffer, the cells can be resuspended on a vortex mixer before spinning again.

If staining in microtiter plate wells, use a centrifuge with plate adaptors. At about 1,500 x g, cells will pellet well in about 2-3 seconds. Removing supernatant fluid after centrifugation can be done by flicking the plates and then blotting once on clean paper towels. More securely, the supernatant can be removed from each well with a glass disposable ("Pasteur") pipette hooked up to suction and a trap. Wash buffer can be added with a multi-channel pipette -- a squirt from this pipette is enough to resuspend a cell pellet if it has not been centrifuged too hard.