Antibody titration protocol using 96 well plates

1. Prepare cells at a final concentration of 2.5×104 per µl in blocking buffer

2. Aliquot 40 µl of cells per sample

If using a viability dye, make sure to include a single stained control for viability (for compensation)

3. Prepare the dilution series

Prepare the concentration so that 10 µl can be added to each sample

4. Add the antibody to the cells and gently mix.

5. Place on ice, in the dark, for 20 minutes

6. Add ~300 µl staining buffer to each well

7. Centrifuge the plate at 900xg

8. Aspirate (or Flick) the solution out of the wells

9. Repeat steps 6-8 two more times

10. Resuspend the sample in ~200 µl final volume

（1）If using viability dye, add to the sample
（2）If sample has to be fixed, resuspend in~200 µl of fixation buffer
（3）If using fixation buffer, cell impermeant viability dyes (PI, 7AAD, DAPI, etc) cannot be used

11. Analyze the cells on the flow cytometer

Collect sufficient events for analysis (10,000 positive events)

12. Export the data and analyze in software of choice

13. Calculate the Staining Index (SI) and plot versus concentration of antibody3

Staining Index (SI) = ((median pos-median neg) / ((84%neg-median neg)*0.995)