Secreted proteins by definition are outside the cell. To trap the protein inside the cell protein transport inhibitors, which interfere with the function of the Golgi apparatus, are used. Cells are then fixed and gently permeabilized before staining with fluorchrome conjugated antibodies.It is important to note that prior stimulation with agents such as PMA/ionomycin is usually required for detection of cytokines.1
The optimal choice of stimulant and protein transport inhibitor will vary by sample type and application. Please consult the literature for best options.
- Activating agent such as PMA (50 ng/ml)/Ionomycin (1 μg/ml) or LPS (100 ng/ml)
- Brefeldin A Solution (1,000X) (Cat. No. 2703005) or Monensin Solution (1,000X) (Cat. No. 2703505)
- Cell Staining Buffer (Sony Cat. No. 2701005)
- Antibodies to cell surface markers conjugated to fluorochromes (if desired)
- Fixation Buffer (Sony Cat. No. 2704005)
- Antibodies to intracellular proteins directly conjugated to fluorochromes
- Intracellular Staining Permeabilization Wash Buffer (10X) (Sony Cat. No. 2705010). It is especially important to include negative controls such as isotype controls as intracellular staining often has higher backgrounds.
- Deionized water (DI)
- 15 or 50 mL conical centrifuge tubes
Stimulate cells for approximately 4-6 hours depending on application. Add Monensin or Brefeldin A to block protein transport.
- Harvest and prepare cells according to appropriate protocol for surface staining.
- Centrifuge cells at 350 x g for 5 min.
- Discard supernatant and resuspend in an appropriate volume of Cell Staining Buffer so that the final cell concentration of cells is 5 – 10 x106 cells/mL.
- Add the appropriate amount of labeled primary antibody to cell surface markers. If the primary antibody needs to be diluted use Cell Staining Buffer. Alternatively all cell surface markers can be stained along with intracellular ones. However, the epitopes may be altered or destroyed by fixation and permeabilization.
- Incubate for 15 – 60 minutes on ice in the dark. Incubation times should be optimized for best signal to noise ratio.
- Fill tube with Cell Staining buffer, centrifuge at 250 x g for 5 min., discard supernatant, and repeat.
- After the last wash discard the supernatant (there will be approximately 100 μl residual). Gently vortex to break up the pellet.
- Add 0.5 ml Fixation buffer and incubate for 20 min. at room temperature in the dark.
- Remove fixative by washing cells once as described in step 6. At this point cells can be resuspended in cell staining buffer and stored at 4°C in the dark (short term).
Permeabilization of Cells
- Dilute 10X Intracellular Staining Perm Wash Buffer to 1X with DI water.
- Resuspend cells fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g, RT for 5-10 minutes.
- Repeat wash twice.
- Resuspend cells in residual Intracellular Staining Perm Wash Buffer (~100 μl) by gentle vortexing to disrupt pellet. Add conjugated antibodies to intracellular antigens (isotype controls, or the combination of intracellular and surface markers) at the recommended/optimized concentration. Incubate in the dark for 20 -60 min. at RT.
- Wash twice in 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at at 350 x g, RT for 5-10 minutes.
- Resuspend in 0.5 ml Cell Staining Buffer and analyze along with the relevant controls.
1Foster, Barbara, et al. "Detection of intracellular cytokines by flow cytometry. " Current protocols in immunology (2007): 6-24.