Flow Cytometry Protocol For Intracellular Proteins
Triton X-100 Method
I. Preparations:
- Fixative solution: 4% Paraformaldehyde in 1x PBS
- Permeabilization solution: 0.5% Triton X-100 in 1x PBS
- Antibody dilution buffer: 0.5% BSA in 1x PBS
- Cell preparation: For suspension cells, cells are pelleted by centrifugation at 250 g. For adherent cells, remove the culture medium, Cells are trypsinized and dissociated into single cells with trituration before centrifugation.
II. Cell fixation and permeabilization:
1. Remove the supernatant, add 100 µL of freshly prepared fixative solution to the tube, and dissociate the pellet into individual cells using a pipette to avoid cross-linking among cells.
2. Fix the cells at room temperature for 20 min.
3. Wash the cells by adding 1 mL of 1x PBS, pellet the cells by centrifugation at 250 g.
4. Discard the supernatant in a biohazard container, resuspend the cells in 100 µL of the permeabilization solution.
5. Permeabilize the cells on ice for a 10 min.
6. Wash the cells by adding 1 mL of 1x PBS, pellet the cells by centrifugation at 250 g.
III. Immunostaining:
7. Discard the supernatant in a biohazard container, resuspend the cells in 100 µL of the primary antibody diluted in the antibody dilution buffer at the recommended dilution rate.
8. Incubate the primary antibody with the cells for 1 hr at room temperature or 4 ℃ overnight.
9. Wash the cells by adding 1 mL of 1x PBS, pellet the cells by centrifugation at 250 g.
10. Discard the supernatant, resuspend the cells in 100 µL of the fluorochrome-conjugated secondary antibody diluted in the antibody dilution buffer at the recommended dilution rate.
11. Incubate the secondary antibody with the cells for 30 min at room temperature. Protect the tube from light.
12. Wash the cells by adding 1 mL of 1x PBS, pellet the cells by centrifugation at 250 g.
13. Discard the supernatant, add 200 µL of 1X PBS, and analyze on a flow cytometer.
