Incorrect Positive/Negative Cell Population Ratios (Flow Cytometry (Controls))

Question

Accepted Answer

The measured ratio of positive to negative cells for a given marker appears inaccurate or inconsistent. Background signals are not correctly measured, leading to improper gating and incorrect population quantification. Common causes: 1) Improper controls used, failing to account for different sources of background signal 2) Incorrect instrument gain (photomultiplier tube voltages) settings not optimized for the fluorochromes used 3) Laser alignment, laser time delay, or sensitivity issues not checked periodically 4) Inappropriate gate boundaries set between positive and negative expression events Solutions: 1) Use appropriate experiment-associated controls (single-stain, FMO, isotype, or biological) to identify main source of background 2) Set instrument gain properly for multicolour experiments by measuring spillover and determining required compensation 3) Perform periodic quality control checks of instrument optics, electronics and fluidics using BD CS&T beads with BD FACSDiva Software 4) Use biological controls (e.g., unstimulated samples) to set relevant boundaries between positive and negative expression when appropriate