Abstract

Setting the PMTs voltages is a
crucial part of any flow cytometry experiment. Recently there has been a lot of
debate on how to set voltages to achieve the best separation between negative
and positive cells and have the minimum spreading error with people proposing
different methods. In this work, we compared three ways to set up voltages:
voltage optimisation, 2.5x standard deviation from the electronic noise and
setting the unstained control cells within the second decade of the graph. We
then calculated the compensation matrix and the spreading matrix for all the
different methods.

Methods: PBMC were stained for
with a anti CD4 antibody all 28 fluorescent parameters 10,000 events per
samples were acquired on the BD Symphony A5 cytometer. Compensation and
spreading matrix were calculated using Flowjo version 10 for all the three
methods, staining index was also calculated.