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 MDS/RRDA, 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% NaN 3 ). Suspend the pellet from the final wash in 50 microliters FACS buffer per each analysis 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.
• Wash once with PBS containing 0.1% saponin, remove supernatant and resuspend the cell pellet with 0.5 ml 2% formaldehyde solution.
• Samples should be at a minimum concentration of 5 X 10 6 cells/ml
• Send to MDS for FACS analysis