Protocols

Immunofluorescent Staining of Human Red Blood Cells (Surface Staining)

1. Add 1 µl whole blood into each sample tube containing 100 µl staining buffer (PBS with 1%FBS, 0.09% sodium azide).
2. Add 20 µl of properly diluted antibody or isotype control to the sample tubes. Mix gently.
3. Incubate at room temperature (RT) for 20-30 minutes.
4. Wash with 2 ml of staining buffer at 200 x g for 5 minutes.
5. Aspirate the supernatant.
6. For purified and biotin conjugate: add 50 µl of diluted secondary reagent, incubate at RT for 15-20 minutes. For direct conjugates proceed to step 8.
7. Repeat steps 3 & 4.
8. Add 0.5 ml of staining buffer to each tube.
9. Proceed to flow cytometric analysis.

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