Phosphorylation Artifacts from Pre-Staining Surface Antibodies (Flow Cytometry (Paraformaldehyde Fixation))

Question

Accepted Answer

Intracellular phosphorylation signals are artifactually elevated or altered when surface antibodies are added before fixation. Antibody binding to surface antigens triggers unwanted intracellular signaling cascades that confound phospho-flow results. Common causes: 1) Surface antibody binding activates receptor-mediated signaling pathways 2) Antibody cross-linking of surface antigens induces phosphorylation cascades 3) Time lag between antibody addition and fixation allows signal transduction 4) Fc receptor engagement by antibodies triggers non-specific phosphorylation events 5) Temperature-dependent signaling continues during surface staining incubation Solutions: 1) Fix cells BEFORE adding surface antibodies when analyzing phospho-targets 2) Use BioLegend True-Phos Perm Buffer protocol with fixation preceding all staining 3) Work on ice (4°C) to minimize signaling if surface staining before fixation is unavoidable 4) Minimize time between surface antibody addition and fixation (<5 minutes) 5) Use Fab fragments instead of whole antibodies to reduce cross-linking effects 6) Validate that surface markers do not alter phosphorylation of targets of interest