We first determined optimal channels (per marker) based on antigen abundance. Next, we determined antibody concentration by testing five different antibody concentrations in a serial dilution. Depending on the nature of the marker, healthy cells were either unstimulated or stimulated with an appropriate biological stimulus, such as PHA or PMA. Finally, spillover was assessed by looking at signals in other channels. To confirm the panel works to detect all major immune cell populations, frequencies of all major immune cell lineages and subsets were measured in PBMCs from 8 healthy subjects.
This is described in finer detail in the Antibody validation section beginning on page 16 of our Panel Validation Whitepaper. Panel Validation Whitepaper