Fluorescent cell barcoding for multiplex flow cytometry

Peter O. Krutzik*, Matthew R. Clutter, Angelica Trejo, Garry P. Nolan

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

78 Scopus citations


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.

Original languageEnglish (US)
Article number6.31
JournalCurrent Protocols in Cytometry
Issue numberSUPPL.55
StatePublished - Jan 2011


  • Barcode
  • Dye
  • Flow cytometry
  • Fluorescence
  • High throughput
  • Multiplex

ASJC Scopus subject areas

  • Medical Laboratory Technology
  • Biochemistry
  • Histology


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