Intracellular Cytokine Staining

 

Solutions:

STAINING BUFFER

PBS, pH 7.4-7.6

2% heat inactivated BCS

0.2% sodium azide

 

FIXATION BUFFER (4% paraformaldehyde)

14 ml 10X PBS

10.8 ml 37.5% formaldehyde

75.2 ml 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 1E6 cells per tube.

2. If necessary, stain for surface markers as per usual FACS protocol.

3. Wash cells with PBS

4. Suspend cells in 0.5 ml PBS and 0.5 ml fixation buffer. Gently vortex and incubate at room temperature for 20 minutes

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.

5. 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 titration.

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 titration. 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 300-500 mL staining buffer and analyze by FACS

 

Notes:

  • Commercial fixative and permiabliziation solutions are available if preferred.
  • Depending on cytokine localization, stronger permeabilizing detergents such as Triton-X or NP-40 may be appropriate (0.1 to 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

Abcam – Intracellular Staining http://www.abcam.com/ps/pdf/protocols/flow_intracellular_staining.pdf