USF Health


Criticizing Flow Cytometry Data by M. Roeder

Questions to ask yourself when you are reading/criticizing FACS data (including your own!)

  • What were the controls?
  • Did the authors use isotype controls? Are isotype controls valid in their experiments?
  • Is the data compensated properly? Look for "diagonals" and over-compensated data.
  • If the graphs are dot plots, are they meaningful? (Is there a "black" area which does not allow for visual density estimation?)
  • If the graphs are contour plots, are they meaningful? How did the authors select the contour levels?
  • If the authors are using antigen density information: Are they using saturating reagents? Did they carefully control their staining protocol? How did they calculate the antigen density (fluorescence): mean, median, geometric mean? Is the data unimodal?
  • If they used a "lymphocyte" gate, is this appropriate for the experiment? i.e., activated cells may be excluded because they are too large.
  • Are their calculations based on channel numbers or fluorescence values (this is relevant if the data was collected with logarithmic amplification, which is virtually always the case).
  • Are they trying to estimate % positive cells for dim antigens-where even if 100% of the cells are positive, the population does not entirely rise above isotype controls?
  • Are they using rectangular gates on populations which cannot be purely identified by rectangular gates?
  • Are they using quad-stats based on autofluorescence? If based on isotype controls, did they specifically use background based on using the isotype with all other antibodies present?
  • For sorting experiments, did they report % purity? Best if they show it!
  • For sorting experiments, do they show the sort gates?
  • When they are doing progressive gating (for instance, in a four-color experiment), do they show examples of how they set the gates?
  • Are they basing their whole experiment on cell-percentages where absolute numbers are the crucial measurement?
  • Are they excluding dead cells?
  • Do you see evidence of non-specific staining (diagonals!)?
  • For lymphocyte analysis, did they properly exclude monocytes? (Look for diagonals!)
  • Did they report in the materials and methods: the instrument, the analysis software, the filters used?

Diagonals: the bane of FACS analysis. 99% of the time, seeing a cluster of events on a 45-degree diagonal, especially one which intersects the origin, is evidence of artifact. Potential artifacts leading to diagonal data: undercompensation, nonspecific staining, autofluorescence.