- Flow Buffer (PBS+2%FBS)
- If using adherent cells: trypsinize, scrape, use Cellstripper (Mediatech Cellgro,Cat#25-056- Cl), or treat with EDTA to get single cell suspension. For cells in suspension, harvest by centrifugation. Centrifuge cells at 350 x g for 5 minutes.
- At least to 1 x 106 cells will be needed for each sample (i.e. 14 x 106 for 7 samples). Transfer the required number of cells to a fresh 15 ml centrifuge tube. Bring the tube to 15 ml with Flow Buffer and invert several times. Centrifuge cells at 350 x g for 5 minutes.
- Resuspend cells in flow buffer so that final volume is 200 ul per sample. Add 200 ul extra to total volume to give allowance. Distribute cells to microcentrifuge tubes.
• You may include a blocking step here. Blocking Fc receptors may help reduce nonspecific immunofluorescent staining. Pre-block cells with excess irrelevant purified Ig from the same species or same isotype as your antibody used for staining.
- Add primary antibody at desired concentration/dilution. An appropriate starting concentration is 4 ug per 1 x 106 cells. Controls should always include an unstained sample, as well as primary alone and secondary alone.
- Incubate cells with primary antibody for 30-60 min in the dark.
- After incubation, wash each sample with 1 ml flow buffer. Centrifuge for 3 minutes at 350 x g for 5 minutes and aspirate buffer. Repeat for total of two washes. If using direct-labeled fluorescent antibody, proceed to step #9.
- Resuspend each pellet in 200 ul flow buffer. Add secondary antibody at desired concentration/dilution. Appropriate dilution is 1:50 or 2 ug total. Incubate 30-60 min @ 4°C in the dark.
- Repeat wash step #6 for a total of two times.
- Resuspend cells in either 200-500 ul flow buffer.
- Transfer cells to the appropriate tubes for flow analysis.