Holden T. Maecker Author Answered Apr 15, 2022
Immunity, Transplantation & Infection, School of Medicine, Stanford University, Stanford
Yes, actually 20 ul antibody cocktail + 50 ul cells = 70 ul total staining volume. We don't check cell numbers, but from 200 ul whole blood, you can expect 500,000-800,000 total leukocytes (including granulocytes). If we do the assay on PBMC, we start with at least 500,000 cells. So 300,000 is a bit low, given losses with washing and acquisition. It would be best to barcode the samples (Fluidigm now sells barcoding kits) and combine them prior to staining, to minimize the loss. We now routinely do barcoding in the context of this assay, as it also saves antibody and increases reproducibility of results across samples that are combined.