Flow Cytometry Laboratory

Intracellular Labeling Protocol- Foxp3 eBiosciences Buffer Kit

Materials

First Wash Buffer (FWB)
450 ml PBS
50 ml ACD
2.25 ml 20% sodium azide in PBS
10 ml horse serum Second Wash Buffer (SWB) FWB without horse serum

eBiosciences Foxp3 Buffer Kit (Cat. No. 00-5523-00)
10X Permeabilization buffer
Fixation/Permeabilization diluent
Fixation/Permeabilization concentrate Perm/Wash solution: dilute 10X Permeabilization buffer 1:10 with distilled water. Make up just before use.

Fix/Perm solution: Dilute 1 part of Fixation/Permeabilization concentrate with 3 parts of Fixation/Permeabilization diluent. Make up just before use.

Protocol

1. Add monoclonal antibodies to a 96 well V bottom plate for surface labeling. Monoclonal antibodies are typically used as 50 μl of 15 μg/ml stock per well.  Place plate on ice.
2. Transfer 1-2 million cells in 100 μl FWB into each well.  
3. Label surface epitopes using standard protocols.
4. Add 200 μl Fix/Perm solution per well.
5. Incubate cells for 30-60 minutes at 4˚ C in the dark.
6. Pellet cells at 2000 rpm for 3 minutes, and aspirate buffer.
7. Vortex plate at low speed to resuspend cells.
8. Wash 2 times with Perm/Wash solution.

Note:   After this step, cells can be resuspended in SWB and stored overnight at 4˚C .  Before proceeding to intracellular antibody incubation, cells must be washed 2 times with Perm/Wash solution.

9. Resuspend cells in 200 μl Perm/Wash solution.
10. Add 50 μl anti-FoxP3 antibody diluted in Perm/Wash solution to each well.
11. Incubate at 4˚ C in the dark for at least 30 minutes.
12. Centrifuge 3 minutes at 2000 rpm at 4˚C  to sediment cells.
13. Aspirate supernatant and vortex at low speed. 
14. Wash 2 times with 200 μl Perm/Wash solution per well.
15. Resuspend cells in 200 μl Perm/Wash solution
16. Add 50 μl of secondary antibody diluted in Perm/Wash solution per well.
17. Incubate at 4˚C in the dark for at least 30 minutes.
18. Centrifuge 3 minutes at 2000 rpm at 4˚C  to sediment cells.
19. Aspirate supernatant and vortex at low speed. 
20. Wash 2 times with 200 μl Perm/Wash solution per well.
21. Resuspend in 200 μl SWB and view on FC.