UNIT 6.31 Fluorescent Cell Barcoding for Multiplex Flow Cytometry

  1. Peter O. Krutzik,
  2. Matthew R. Clutter,
  3. Angelica Trejo,
  4. Garry P. Nolan

Published Online: 1 JAN 2011

DOI: 10.1002/0471142956.cy0631s55

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Krutzik, P. O., Clutter, M. R., Trejo, A. and Nolan, G. P. 2011. Fluorescent Cell Barcoding for Multiplex Flow Cytometry. Current Protocols in Cytometry. 55:6.31:6.31.1–6.31.15.

Author Information

  1. Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, California

Publication History

  1. Published Online: 1 JAN 2011
  2. Published Print: JAN 2011


Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10- to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed. Curr. Protoc. Cytom. 55:6.31.1-6.31.15. © 2011 by John Wiley & Sons, Inc.


  • flow cytometry;
  • multiplex;
  • barcode;
  • fluorescence;
  • dye;
  • high throughput