Joris van Dongen

136 Chapter 6 19. Centrifuge the sample at 800 xg at 8°C for 10 min. 20. Remove supernatant. 21. Resuspend cell pellet in lysis buffer and place the sample on ice for 5 min. 22. Centrifuge the sample at 800 xg at 8°C for 10 min. 23. Remove supernatant. 24. Repeat step 19. and 21. till all erythrocytes are disrupted and the red color has disappeared. 25. Resuspend cell pellet in FACS buffer and divide cells inmultiple tubes. The number of tubes depends on the number of subset of CD markers used. Additionally, one tube will function as a blanc control and for each fluorophore used, an extra tube will be used for the fluorophore specific isotype control (see Note 4). 26. Centrifuge cells at 300 xg at 4°C for 5 min. 27. Resuspend cell pellet in 100 μL FACS buffer. 28. Keep one tube of cells unlabeled and put on ice in the dark for 30 min. This tube functions as a blank control to set up the flow cytometer. 29. Incubate a tube of cells on ice with 5 μL of the preferred fluorophore-conjugated antibodies (1:20) in the dark for 30 min. (Table 1. and see Note 5). 30. Incubate the other tubes of cells on ice with 5 μL of the specific fluorophore isotype control (1:20) in the dark for 30 min. 31. Wash samples with 2 ml FACS buffer. 32. Centrifuge cells at 300 xg at 4°C for 5 min. 33. Remove supernatant. 34. Repeat step 10. till 12. three times. 35. Resuspend cell pellet in 300 μL FACS buffer. 36. Proceed to FACS analysis.