Pre-sort efficiency test with TMB and HRP for 384 well plate

1. Aliquot 1 µl TMB into a 384-well plate.
2. To 300 µl single cell suspension (ideally in PBS) or PBS with one droplet of FACS beads add 100 µl of a 250 µg/ml HRP solution. From older stocks of HRP increase the final concentration from 62.5 µg/ml to 100 µg/ml.
3. Set up the FACS sorter, including all sorting alignment. Check the calibration by sorting 50 cells, from the HRP solution on the lid of few wells of the prepared plate and make sure the droplets are in the centre of the wells. If not, make necessary alterations and re-test.
4. Sort 1 cell (or FACS bead) into every well of the previously prepared TMB plate.
5. Check how many wells in the plate have turned blue, this might take some minutes, and should be >90 % for good sorting efficiency.

References

Rodrigues, Olivia Roos, and Simon Monard. 2016. “A Rapid Method to Verify Single-Cell Deposition Setup for Cell Sorters.” Cytometry Part A 89 (6): 594–600. https://doi.org/10.1002/cyto.a.22865.