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
- 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
- Are they basing their whole experiment on cell-percentages
where absolute numbers are the crucial measurement?
- Are they exluding 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 artefact. Potential
artefacts leading to diagonal data: undercompensation, nonspecific