Intracellular Staining

Intracellular Staining of Human Red Blood Cells

1. For each sample to be stained, fix 10 µl of whole blood in 1 ml cold 0.05% glutaraldehyde for 10 minutes at room temperature. Vortex after addition of cells.
2. Wash once with 5 ml staining buffer (PBS with 1%FBS, 0.09%NaN 3). Centrifuge at 200 x g for 5 minutes.
3. Resuspend the cell pellet by vortexing in 0.5 ml 0.1% Triton X-100. Incubate for 10 minutes at room temperature.
4. Pellet by centrifugation at 200 x g for 5 minutes and aspirate the supernatant.
5. Resuspend the red blood cell pellet in 0.5 ml staining buffer.
6. Into new tubes, add primary antibody to appropriate concentration in staining buffer to make 100 µl total volume. For example, if the antibody test size volume is 20 µl, add this amount to 80 µl of staining buffer. Add 10 µl of red blood cell suspension to each tube. Incubate for 20 minutes at room temperature (in the dark).
7. Wash with 2 ml of staining buffer at 200 x g for 5 minutes.
8. For purified and biotin conjugated antibodies: add 50 µl of diluted secondary reagent, incubate at RT for 15-20 minutes. For direct conjugates proceed to step 10.
9. Repeat step 7.
10. Add 0.5 ml of staining buffer to each tube.
11. Proceed to flow cytometric analysis.

Data Acquisition Note: Setting FSC and SSC detectors to log mode provides better resolution compared to linear mode.