Multiple Cell Populations When Expecting Single Population (Flow Cytometry (Troubleshooting))

Question

Accepted Answer

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. Common causes: 1) Cell doublets (aggregates of two cells) present in sample, appearing as population at approximately 2x fluorescence intensity 2) Multiple cell types present in heterogeneous sample, each expressing target protein at different levels 3) Inadequate cell separation or purification before staining, leaving mixed populations Solutions: 1) Mix cells gently using pipette before staining and again immediately before running on cytometer to dissociate doublets 2) Filter cells through 30 μm nylon mesh to remove clumps and doublets 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