Introduction
Flow Cytometry is a technology that measures and analyzes several physical characteristics of single cells. The cells flow in a fluid stream through a beam of laser light. Using Flow Cytometry the following cell characteristics can be determined: cell size, cell granularity, complexity and relative fluorescence intensity.
Flow Cytometry Intracellular and Membrane Staining Protocol
Cell fixation (for membrane protein):
- Suspend cells in 1x PBS buffer and wash them twice with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time. Discard the supernatant.
- Re-suspend the cells in 1 ml of 1x PBS buffer briefly.
- Fix the cells in a final concentration of 4% formaldehyde (or paraformaldehyde) for 20 min at room temperature.
- Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time.
Cell fixation and Permeabilization (for intracellular protein):
- Permeabilize cells by adding 100% cold methanol slowly to pre-chilled cells to a final concentration of 90% methanol before incubating for 30 min on ice.
- Alternatively, fix the cells in a final concentration of 4% formaldehyde (or paraformaldehyde) for 20 min at room temperature. Then incubate the cells in 0.1% Triton X-100 in 1x PBS buffer for 15 min at room temperature.
- Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time.
Immunostaining:
- Blocking: Incubate the cells with 3 ml blocking buffer for 1h at room temperature.
- Add primary antibody at an appropriate dilution and incubate for 1h at room temperature.
- Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time.
- Add diluted secondary antibody (enzyme or fluorescein conjugated or other types) to the cells and incubate for 1h at room temperature.
- Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time.
- Re-suspend the cells in 0.5 ml 1x PBS buffer and analyze the results on a flow cytometer. For DNA staining, re-suspend the cells in 0.5 ml of DNA dye instead; incubate for at least 5 min at room temperature before analyzing the results on a flow cytometer.
Buffers needed:
| Blocking Buffer | 1000 ml |
| Bovine serum albumin | 5.00 g |
| 1x PBS buffer | 1000 ml |
| PBS Buffer | 1000 ml |
| 10 mM Na₂HPO₄ | 1.42 g |
| 1.7 mM NaH₂PO₄ | 0.20 g |
| 140 mM NaCl | 8.19 g |
| Add ddH₂O to 1000 ml | |
| Adjust to pH 7.4 | |