Collect blood (75 microliters) into 1ml PBS containing 5 microM EDTA (10 microliters of 0.5 M stock) and mix immediately to prevent clotting. Keep tubes on ice.
Remove RBCs from samples. This can be accomplished by several means. At The Jackson Laboratory, RBCs are lysed using either Gey's solution or a buffered ammonium chloride (ACK) solution. (Alternatively, Becton-Dickinson sells a product called "FACS lysis buffer" that is used after the staining protocol to lyse RBCs and fix the cells.)
Cells should be washed 2-3x with FACS buffer (PBS supplemented with either 1% BSA or 5% FBS and containing 0.05% NaN3). Suspend the pellet from the final wash in 50 microliters FACS buffer (or more if more than one analysis is to be done on a single sample - up to three separate staining reactions can be set up from a single sample).
Add 50 microliters of cell suspension to 10 microliters of antibody solution and mix gently. You will need to determine the proper concentration for each antibody used.
Incubate for 30 minutes on ice.
Wash cells 2-3x with FACS buffer and suspend in 200-300 microliters FACS buffer for analysis.
For live/dead discrimination, add 10 microliters propidium iodide (PI) solution (stock=10 micrograms/ml). If fixing cells before analysis, do not add PI.
