High Signal in Negative Cell Populations (Flow Cytometry (CST Guide))

Question

Accepted Answer

Off-target cell populations such as monocytes show unexpectedly high fluorescence signal. Non-specific staining obscures true positive populations. Common causes: 1) Fc surface receptors on monocytes or other cells binding Fc portion of primary or secondary antibodies 2) Excessive primary or secondary antibody causing non-specific binding 3) Incomplete washing between antibody incubation steps leaving unbound antibody Solutions: 1) Block cells with Bovine Serum Albumin, Fc receptor blocking reagents, or normal serum from same host as primary/secondary antibody prior to staining 2) Include secondary-antibody-only control to determine if background originates from primary or secondary antibody 3) Perform additional wash steps between antibody incubations to remove unbound antibody