FACS Staining

• If adherent cells, trypsinize, scrape, or treat with EDTA to get single cell suspension.
• Count cells, at least 2 x 106 cells will be needed for each sample.
• Spin cells down (5 min @ 100 x g in tabletop centrifuge).
• Resuspend cells to 2 x 106 cells/200 mL in 2% FBS/PBS (FACS buffer).
• Transfer 200- mL volumes to individual 1.5 mL microcentrifuge tubes.
• Add 10 µL 1° Ab at the desired dilution to appropriate tubes. Incubate 1 hr at 4°C.
  
• Optimal concentration for each antibody will need to be determined by the end user.
• Add 1 ml FACS buffer and spin down. (3 min @ 800 x g in Eppendorf microcentrifuge)
• Aspirate the supernatant.
• Wash with 1 ml FACS buffer, spin down again
• Aspirate the supernatant.
• Add 2° Ab at desired concentration in 100 mL of FACS buffer + 1% BSA to appropriate tubes.
• Incubate at 4°C for 30 minutes.
• Repeat steps 7-10.
• Resuspend in 500 mL FACS buffer.
• Transfer into 12 x 75 mm polystyrene FALCON # 2024 snap-cap, 5 ml test tubes.
• If fixing cells before analysis, do not add propidium iodide.
• Samples should be at a minimum concentration of 5 X 10 6 cells/mL
  
• Send to MDS for FACS analysis