Foxp3 / Transcription Factor Staining Buffer Kit Cat. No. TNB-­0607-­KIT

Kit Includes:

Other Materials Required

  • Flow Cytometry Staining Buffer (Stain Buffer) (1X PBS with 2% FBS, 0.09% Na­Azide)

Buffer and Solution Preparation

  1. Prepare fresh Transcription Factor Fixation/Permeabilization working solution by diluting Transcription Factor Fixation/Permeabilization Concentrate (1 part) with Transcription Factor Fixation/Permeabilization Diluent (3 parts). You will need 1 mL of the Transcription Factor Fixation/Permeabilization working solution for each sample.
  2. Prepare a 1X working solution of Flow Cytometry Perm Buffer by diluting the 10X concentrate with distilled water prior to use. You will need 3­-5 mL of Flow Cytometry Perm Buffer working solution for each sample.

Experimental Procedure

  1. Aliquot cell samples to tubes in a volume and at a cell concentration suitable for staining.
  2. Stain cell surface antigen(s) with the recommended optimal concentration of fluorochrome labeled antibodies.
  3. Incubate for 20­-30 minutes at 4°C or room temperature. Samples should be protected from light.
  4. Wash cells with 1­-2 mL Stain Buffer.
  5. Centrifuge samples at 300-­400 x g at room temperature for 5 minutes, discard the supernatant.
  6. Vortex sample (<5 seconds) to completely dissociate the cell pellet.
  7. Add 1 mL Transcription Factor Fixation/Permeabilization working solution to each tube and pulse vortex (< 5 seconds).
  8. Incubate at 4°C or room temperature for 30­-60 minutes in the dark.
  9. Centrifuge samples at 300-­400 x g at room temperature for 5 minutes, discard the supernatant.
  10. Wash cells with 1­-2 mL Flow Cytometry Perm Buffer working solution.
  11. Centrifuge samples at 300­-400 x g at room temperature for 5 minutes, discard the supernatant.
  12. [Optional] Block with 2% normal mouse/rat serum by adding 2 µL directly to the cells. Incubate at room temperature for 15 minutes.
  13. Without washing, add the recommended amount of fluorochrome­-conjugated antibody for detection of intracellular antigen to cells and incubate in the dark at room temperature for at least 30 minutes.
  14. Wash cells with 1-­2 mL Flow Cytometry Perm Buffer working solution.
  15. Centrifuge samples at 300-­400 x g at room temperature for 5 minutes, discard the supernatant.
  16. Wash cells with 1­-2 mL Stain Buffer.
  17. Centrifuge samples at 300­-400 x g at room temperature for 5 minutes, discard the supernatant.
  18. Resuspend stained cells in an appropriate volume of Stain Buffer and acquire data on a flow cytometer.

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