Extracellular Staining

1. Process and count cells in FACS buffer (0.2% BSA in PBS, filtered).

2. Plate 1x106 cells in 96-well round bottom plate. Spin at 1200 rpm for 5-10 minutes at 4’C. Dump.

3. Prepare FC block in 1:100 dilution with FACS buffer. Add 100ul to each well. Resuspend.

4. Leave on ice for 20 minutes.

5. Spin at 1200 for 10 minutes at 4’C with brakes on. Dump.

6. Add 100ul of FACS buffer for unstained samples and 100uL of antibody to samples. Make sure it is done in light sensitive environment.

7. Wrap in tin foil and incubate for 30 minutes on ice

8. Spin at 1200 rpm for 10 minutes at 4’C with brakes on. Dump

9. Wash with 100uL of FACS buffer, resuspend, and spin again. Dump

           Note: Proceed to intracellular staining method below if necessary. Otherwise                         continue onto Step 10.

 10. Prepare 1% PFA with PBS and add 100uL per well.
 11. Cover tightly with tin foil and store at 4’C.
 12. Following day, transfer 100uL of PFA into FACS tube. Keep on ice.
 13. Spin at 1000 for 2 minutes at 4’C with brakes on.

 

Intracellular Staining

1. Add 150uL of BD Cytofix/Cytoperm per well. Keep on ice in dark for 20 minutes.

2. Spin at 1200 rpm for 10 minutes at 4’C with brakes on. Dump

3. Prepare 1x BD Perm/Wash solution (from 10x solution) with ddH2O. Resuspend with 100uL per well.

4. Spin at 1200 rpm for 10 minutes at 4’C with brakes on. Dump.

5. Prepare intracellular antibody in BD Perm/Wash (not FACS buffer). Add 100uLof antibody to samples. Wrap in tin foil and ice for 20 minutes.

6. Spin at 1200 rpm fro 10 minutes at 4’C with brakes on. Dump.

7. Wash with 100uL of BD Perm/Wash. Resuspend, spin and dump.

8. Add 100ul of 1% PFA or add 100uL of FACS buffer if running flow immediately. Cover tightly with tin foil and store at 4’C

Note:

-If making more then one treatment group, make antibody master mixes (i.e. for 12 groups add 13uL:1300uL FC block to PBS…)