Considerations for FACS Gating

To achieve accurate results, it is important to use an optimal gating strategy when assessing cell samples via Flow Cytometry. Factors to consider and incorporate into your gating strategy include:

  • 1) Exclusion of debris
  • 2) Utilizing appropriate fluorophore negative controls
  • 3) Exclusion of non-viable cells
  • 4) Staining with a shared marker (i.e. CD45 leukocyte marker or equivalent pan-population marker) when appropriate
  • 5) Setting up necessary fluorophore analysis plots

Exclusion of debris:
When acquiring data on the flow cytometer, create a Forward Scatter (FSC) versus Side Scatter (SSC) plot and ensure that all the expected cell populations are visible by adjusting the individual FSC and SSC photomultiplier tube settings. This will also necessitate making sure that most of the debris, air bubbles and laser noise (all which should be FSC-low), are removed from analysis by setting and adjusting the FSC Threshold as necessary. Next, insert a region (R1) around your cell populations of interest on this FSC vs SSC plot. This region can be regarded as "Region 1 - FSC vs SSC".

(Typical setup profile for Lysed Human Whole Blood)

2) Utilization of appropriate fluorophore negative controls:
When staining your cells, it is advisable that you split them into two tubes, where one tube has been stained with the fluorophore-conjugated antibody or stain of interest, while the other tube has the appropriate negative control added. An isotype fluorophore can act as a negative control for comparison, which facilitates accurate distinction between the positive signal and negative background fluorescence in your analysis.

3) Exclusion of non-viable cells:
Cells should be stained with a viability dye such as Propidium Iodide (PI) or 7-AAD in order to discern between living and dead cells. First, create a FSC versus Viability stain plot, and then apply the "Region 1 - FSC vs SSC" gate onto it. Afterwards, insert a region around the viable cell populations, this will be "Region 2 - Viable".

4) Staining with a shared marker when appropriate:
While not always necessary, but useful when dealing with rare cell types, it is desirable to stain with a marker that your cells of interest as well as your contaminating cells all express (such as, CD45, which is expressed on all leukocytes). First, create a Forward Scatter (FSC) versus Shared Marker plot and then apply the "Region 2 - Viable" gate onto it. Afterwards, insert a region around the cell populations that express this shared marker, this will be "Region 3 - Shared Marker".

5) Setting up necessary fluorophore analysis plots:
Necessary fluorophore analysis plots can now be created as needed (i.e. FSC vs FITC or FITC vs PE, etc.). Please be sure that "Region 1 - FSC vs SSC", "Region 2 - Viable", and if applicable "Region 3 - Shared Marker", gates are applied onto each of these fluorophore plots. Use the appropriate fluorophore isotype negative controls to help discern the positive signal from the background fluorescence.

Please consult your Flow Cytometer's manual on appropriate compensation procedure, or your Flow Cytometry Analysis Software's documentation for more detailed instructions.

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