Foxp3 Staining Protocol

The following protocol is for intracellular staining of Human Foxp3 using fluorochrome conjugated Foxp3 antibodies (such as clone PCH101 or 236A/E7). Please refer to specific clones for the corresponding protocol. Staining of mouse or rat tissue can be modified slightly with an extended incubation step in the Fixation/permeabilization solution. It is critical to use the Foxp3 Staining Buffer Set (cat. 00-5523). The buffer set is included with all Foxp3 Staining Sets.

We have also determined that this kit is suitable staining for transcription factors (such as T-bet and nanog) and most cytokines. Please contact tech@ebioscience.com for additional information.

Prior to staining, dilute the Fixation/Permeabilization Concentrate (1 part) into the Fixation/Permeabilization Diluent (3 parts) to the desired volume of Fixation/Permeabilization working solution. This buffer should not be stored for more than 1 day. For example: For 12 samples, use 3 ml Fixation/Permeabilization Concentrate and 9 ml Fixation/Permeabilization Diluent. The Permeabilization Buffer is supplied as a 10X concentrate. The 10X stock should be diluted in distilled water to a 1X solution prior to use. The 1X permeabilization buffer should be made fresh before each experiment.

1. Add 100 µl of prepared cells (1x10⁶) to each tube.
2. Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol (http://www.ebioscience.com/ebioscience/appls/FCS.htm).
3. Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
4. Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Fixation/Permeabilization working solution to each sample. Pulse vortex again.
5. Incubate at 4°C for 30 - 60 minutes in the dark.
6. Wash once by adding 2 ml 1X Permeabilization Buffer (made from 10X Permeabilization Buffer) followed by centrifugation and decanting of supernatant.
7. OPTIONAL] Block with 2% (2 µl) normal rat serum in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
8. Without washing after blocking step, add 20 µl fluorochrome conjugated anti-human Foxp3 antibody or isotype control in 1X Permeabilization Buffer and incubate at 4°C for at least 30 minutes in the dark. Please perform further titration for optimal staining in your own assay system.
9. Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
10. Repeat step 9.
11. Resuspend in appropriate volume Flow Cytometry Staining Buffer and analyze on cytometer. Please note that due to the fixation and permeabilization procedure, the FSC/SSC distribution of the cell population will be different than live cells. Therefore the gate and voltages will need to be modified.