I am very new to flow. I stimulated the Healthy PBMC and want to get the stimulate T cell, but I don't know if I should keep the same cell numbers in all wells? So far, I set unstained 250,000 cells, minus-one group 500,000 cells and targeted well 1x10^6 cells .The reason why I am not sure is we are using the CD3/CD28 to stimulate the cells . I wondering should I keep them all in the same number like 500,000 per well or as stated above?