UNIT 6.3 High-Sensitivity Immunofluorescence/Flow Cytometry: Detection of Cytokine Receptors and Other Low-Abundance Membrane Molecules
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Cytometry
How to Cite
Zola, H. 2001. High-Sensitivity Immunofluorescence/Flow Cytometry: Detection of Cytokine Receptors and Other Low-Abundance Membrane Molecules. Current Protocols in Cytometry. 6:6.3.
- Published Online: 1 MAY 2001
- Published Print: JUL 1998
This is not the most recent version of the article. View current version (1 NOV 2004)
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, is about 2,000 molecules of target antigen per cell, which is adequate for most of the widely used leucocyte markers. However, measuring target antigens of low density has proved very difficult indeed. Flow cytometric immunofluorescence is capable of detecting 100 molecules of target antigen per cell in practical applications, provided 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.