High‐Sensitivity Immunofluorescence/Flow Cytometry: Detection of Cytokine Receptors and Other Low‐Abundance Membrane Molecules

Cell marker identification by traditional phenotyping techniques is now considered straight-forward and relatively uncomplicated. In immunofluorescence/flow cytometry, the sensitivity, or detection limit, depends on the reagents, staining, and instrument parameters. The sensitivity of the most commonly used procedures, based on fluorescein-conjugated antibodies, approximately 2000 molecules of target antigen per cell, which is adequate for most of the widely used leukocyte markers. However, measuring target antigens of low density has proven very difficult indeed. Flow cytometric immunofluorescence is capable of detecting 100 molecules of target antigen per cell in practical applications, provided that every step of the staining and analysis procedure is optimized for sensitivity. This level of sensitivity reveals staining not seen using conventional analytical procedures. This unit discusses the underlying principles of high-sensitivity immunofluorescence and provides an excellent series of protocols for the practical detection of as few as 100 target antigen molecules per cell.