Necessary controls:

• Cells only
• 1° Ab only
• 2° Ab only

1. If adherent cells, trypsinize, scrape, or treat with EDTA to get single cell suspension.

2. Count cells, at least 2 x 106 cells will be needed for each sample.

3. Spin cells down (5 min @ 100 x g in tabletop centrifuge).

4. Resuspend cells to 2 x 106 cells/200 μl in 2% FBS/PBS (FACS buffer).

5. Transfer 200-μl volumes to individual 1.5 mL microcentrifuge tubes.

6. 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.

7. Add 1 ml FACS buffer and spin down. (3 min @ 800 x g in Eppendorf microcentrifuge)

8. Aspirate the supernatant.

9. Wash with 1 ml FACS buffer, spin down again.

10. Aspirate the supernatant.

11. Add 2° Ab at desired concentration in 100 μl of FACS buffer + 1% BSA to appropriate tubes.

12. Incubate at 4°C for 30 minutes.

13. Repeat steps 7-10.

14. Resuspend in 500 μl FACS buffer.

15. 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.