Flow Cytometry (FCM) / FACS Antibody Verified by Non-conjugated Antibody Blocking

Flow cytometry is often using a labeled antibody directly detects cell surface or internal antigen. Verified by unlabeled antibody competing binding of the antigen, in flow cytometry, the labeled antibody signal strength will be significantly decreased. One purpose of this verification experiment with unlabeled antibodies is qualifying whether the specificity change after the labelling. Another purpose is to make known that labelled antibody containing few unbound or fall off labelling molecule, which itself is the likelihood of non-specific binding to the specimen.

For example,

Antigen: Human CD146
Antibody: CD146 / MCAM Antibody (FITC), Rabbit MAb (10115-R044-F)
Cell: HeLa
Flow Cyt: Use 10 µl for 10^6 cells
Instrument: BD FACSCalibur

Flow Cytometry / FACS Background

Flow cytometry is a method to evaluate cell membrane proteins and intracellular proteins as well as peptides and DNA. The principle behind FACS is an antigen-antibody reaction, with the antibodies being fluorescently labelled. There are three fluorescent proteins (R-PE, APC, and PerCP) conjugated to antibodies. Flow cytometry quantification is carried out with intercalating color labels (without the antibody). Flow cytometry antibodies are widely used in cell counting, cell sorting, biomarker detection and protein engineering.