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
Staining buffer + 0.5% saponin
3 parts Perm Buffer + 1 part BCS (filtered)
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
- 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% )
- Shimizu Lab/Author Nadine Ottoson http://www.tc.umn.edu/~shimi002/sopicfacs.pdf
- 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