Non-specific Antibody Binding Creating Noise (Flow Cytometry (Controls))

Question

Accepted Answer

Antibody binds non-specifically to cells of interest, resulting in noisy data and elevated background. True marker expression cannot be distinguished from non-specific binding events. Common causes: 1) Test antibody exhibits non-specific binding to cell populations beyond target marker 2) FMO controls cannot measure this background as they lack the antibody being studied 3) Isotype control does not match the background characteristics of the test antibody 4) Factors affecting background (antibody concentration, incubation time, Fc receptor binding) not controlled Solutions: 1) Use isotype controls matching the test antibody isotype and conjugated to the same fluorochrome to measure non-specific binding 2) Ensure isotype control antibody concentration and conditions match the test antibody as closely as possible 3) Recognize that isotype controls do not account for fluorescence spillover from other channels; use with compensation 4) Run multiple control types during assay development to determine if non-specific binding is the main background source 5) Consider optimizing antibody concentration, blocking Fc receptors, or adjusting incubation conditions to reduce non-specific binding