INTRACELLULAR CYTOKINE STAINING

Solutions:

Staining Buffer:

  • PBS, pH 7.4-7.6
  • 2% heat inactivated BCS
  • 0.2% sodium azide

Fixation Buffer (4% paraformaldehyde)

  • 14mL 10X PBS
  • 10.8mL 37.5% formaldehyde
  • 75.2mL dH2O

Perm Buffer

  • Staining Buffer + 0.5% saponin

Superperm Buffer

  • 3 parts Perm Buffer + 1 part BCS (filtered)

Staining Protocol

After harvesting and any stimulation procedures, incubate cells on ice for 20 minutes. Keep cells on ice until fixation.

  1. Use a minimum of 1x106 cells per tube
  2. If necessary, stain for surface markers as per usual FACS protocol
  3. Wash cells with PBS
  4. Suspend cells in 0.5mL PBS and 0.5mL fixation buffer. Gently vortex and incubate at room temperature for 20 minutes
  5. Optional - To store cells after fixation, spin cells down and repeat fixation step, suspend cells in Staining Buffer and store refrigerated for up to 3 days.
  6. Wash with PBS

All Intracellular Staining steps should be in saponin containing buffers

  1. Wash with Perm Buffer once and then wash once with Superperm Buffer. After fixation, cells can be vortexed in the usual manner
  2. Add primary antibody for intracellular staining. It is recommended to use 2x the normal amount that would be used for surface staining, but you may need to optimize by tritration
  3. Incubate in the dark at room temperature for 30 minutes
  4. Wash with Perm Buffer twice
  5. If necessary, add secondary antibody. It is recommended to use 2x the normal amount that would be used for surface staining, but you may need to optimize by tritration. Secondary PE-conjugated reagents are recommended to get the maximum signal.
  6. Incubate at room temperature for 30 minutes
  7. Wash with Perm Buffer twice
  8. Wash with PBS once
  9. Wash with Staining Buffer once
  10. Resuspend in 300-500mL Staining Buffer and analyze by FACS

Notes:

  • commerical fixative and permiabilization solutions are available if preferred
  • Depending on cytokine localization, stronger permeabilizing detergents such as Triton-X or NP-40 may be appropriate (0.1% - 1%)

Adapted From:

  1. Shimizu Lab/Author Nadine Ottoson http://www.tc.umn.edu/~shimi002/sopicfacs.pdf
  2. Think Peptides - Intracellular Cytokine Staining http://www.thinkpeptides.com/PR02TP.pdf
  3. Abcam - Intracellular Staining http://www.abcam.com/ps/pdf/protocols/flow_intracellular_staining.pdf