Fixed-Intracellular Staining for Flow Cytometry

Materials:

  • Flow Buffer (PBS+2%FBS)
  • Permeabilization Bufer (PBS+10% FBS+0.1% Saponin)
  • Fixation Buffer (4% paraformaldehyde)

Procedure:

Note: If staining for both surface and intracellular antigens, first perform cell surface antigen staining as described in Cell Surface Immunofluorescence Staining of Cells for Flow Cytometry, then follow procedure for fixation and permeabilization beginning with step #3.

  1. If using adherent cells: trypsinize, scrape, use Cellstripper (Mediatech Cellgro,Cat#25-056-Cl), or treat with EDTA to get single cell suspension. For cells in suspension, harvest by centrifugation. Centrifuge cells at 350 x g for 5 minutes.
  2. At least to 2 x 106 cells will be needed for each sample (i.e. 14 x 106 for 7 samples). Transfer the required number of cells to a fresh 15 ml centrifuge tube. Bring the tube to 15 ml with Flow Buffer and invert several times. Centrifuge cells at 350 x g for 5 minutes.
  3. In 15 ml centrifuge tube, wash cell with 10 ml of Fixation Buffer. Centrifuge cells at 350 x g for 5 minutes and aspirate media.
  4. Add 10ml of Fixation Buffer and incubate for 15 minutes in the dark.
  5. Centrifuge cells at 350 x g for 5 minutes and aspirate media.
  6. Wash pellet with 10 ml of Flow Buffer and centrifuge cells at 350 x g for 5 minutes and aspirate media.
  7. Wash pellet with 10 ml Permeabilization Buffer and centrifuge cells at 350 x g for 5 minutes and aspirate media.
  8. Re-suspend pellet in 100 ul/1 x 106 cells Permeabilization Buffer. Aliquot 200 ul for each sample into 1.5 ml microcentrifuge tubes.
  9. Add 1° Ab in desired concentration to appropriate tubes. Incubate 30 min in the dark. If using an antibody already fluorescently labeled, proceed to step 13.
  10. After incubation, wash each sample with 1 ml Flow Buffer. Centrifuge for 3 minutes at 350 x g. Aspirate the supernatant. Repeat for total of two washes.
  11. Re-suspend samples in 200 ul of Permeabilization Buffer.
  12. Add 2° Ab and incubate for 30 minutes in the dark.
  13. After incubation, wash each sample with 1 ml Flow Buffer. Centrifuge for 3 minutes at 350 x g. Aspirate the supernatant. Repeat for total of two washes.
  14. Re-suspend each sample in 200 ul Flow Buffer. Samples are now ready for analysis.