September 22, 2011  |  Flow Cytometry  |  By  |  2 Comments

Deconvolute, Decode, Decipher! How to Split, Tag, and Analyze Your Barcoded Data on Cytobank

You may have heard about Fluorescent Cell Barcoding, a flow cytometry technique that allows researchers to answer a larger number of questions with the same amount of antibody, as compared to standard flow cytometry experiments [1,2]. We’ve prepared a few resources to help you learn about, perform, and analyze barcoding experiments.


How does barcoding work? In the barcoding step, samples treated under different stimulation conditions are labeled with concentrations of dye that increase at a defined interval. The use of this dye to barcode effectively means that one cytometer channel is taken up for this code. The distinctly stimulated and labeled samples are then combined into one tube and stained with antibodies against targets of interest. This single tube is then run on a flow cytometer and data are collected for analysis. The most common approach is to barcode different stimulation conditions; however, barcoding can be applied to any distinct populations, such as patient samples or different time points of a stimulation condition.

(Adapted from Krutzik and Nolan, Nature Methods 2006)

Performing the Experiment and Analysis on Cytobank

We’ve prepared a video tutorial that walks you through how to work with barcoded data on Cytobank (skip to 2:01 to jump straight to Cytobank analysis, if you wish). You can follow along by cloning your own copy of Experiment #8938. In this experiment, human PBMCs were stimulated with four stimuli, and two unstimulated conditions were included, resulting in the need to barcode such that these six sample types can be distinguished when combined into one tube. Two barcoding dyes were used to create a 3 by 2 barcoding matrix. We drew gates to define the barcoded populations, exported the gated data into separate FCS files, and proceeded with analysis as usual in Cytobank. In the exporting step, Cytobank computationally separates the barcoded cells into different files, as if you had never barcoded them to begin with. Then files are easily analyzed in standard flow cytometry packages, including Cytobank. Splitting off the barcoded cells into separate files dramatically reduces the processing power (and time) required to perform analysis in any software package.

For a condensed summary of how to work with barcoded data on Cytobank, see our text-based barcoding tutorial.

Additional Information

Want to learn more about the nuances of barcoding, such as how to choose barcoding dyes? Visit our Barcoding FAQ documentation. As always, you can submit a support ticket to request additional help by email or WebEx.


[1] Krutzik PO, Clutter MR, Trejo A, Nolan GP. Fluorescent cell barcoding for multiplex flow cytometry. Curr Protoc Cytom. 2011. Chapter 6:Unit 6.31.

[2] Krutzik PO and Nolan GP. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nature Methods. 2006. 3(5):361-8.

– Angela