FACS Analysis Using Peripheral Blood Cells
- 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.