Multiple Cell Populations When Expecting Single Population

Symptom

Flow cytometry plots display two or more distinct cell populations where only one homogeneous population was expected. A second population often appears at approximately twice the fluorescence intensity of the primary population.

1. 1 Cell doublets (aggregates of two cells) present in sample, appearing as population at approximately 2x fluorescence intensity
2. 2 Multiple cell types present in heterogeneous sample, each expressing target protein at different levels
3. 3 Inadequate cell separation or purification before staining, leaving mixed populations

1. 1 Mix cells gently using pipette before staining and again immediately before running on cytometer to dissociate doublets
2. 2 Filter cells through 30 μm nylon mesh to remove clumps and doublets
3. 3 Check expected expression levels from all cell types in sample; perform adequate cell separation using isolation kits if necessary; use FSC-A vs FSC-H or SSC-A vs SSC-H gating to identify and exclude doublets during analysis

Related Video (3)

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"Covers the full flow cytometry workflow from sample prep through analysis with an explicit troubleshooting component where doublet artifacts would be addressed."

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"Hands-on protocol including sample preparation and parameter adjustment, the steps where doublet formation and single-population validation are practically handled."

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"Demonstrates gating, scatter plots, and histogram analysis on the instrument, directly relevant to recognizing and excluding doublet populations on flow plots."