Retain the population being measured
Detached and adherent cells may differ in state after treatment. If the aim is to assess the whole culture, discarding cells in the supernatant changes the population measured. Decide whether to combine fractions and apply the same rule to every group.
Control handling-time differences
Record collection, staining completion and acquisition times. Use consistent collection methods and comparable waiting periods. If negative controls also shift with waiting time, review handling and acquisition scheduling first.