Fluorescent Cell Barcoding is a flow cytometry technique that allows you to answer a larger number of questions in an experiment using the same amount of antibody. 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.

View our barcoding video tutorial or text-based tutorial.

Here are some Frequently Asked Questions and their answers:

Q: Can I barcode patient samples to analyze clinical data? Won't the forward and side scatter difference result in indistinguishable barcoding matrix populations?

A: The most common approach is to use barcoding to differentiate differently stimulated samples; however, barcoding can be applied to any distinct populations, such as unique patient samples or different time points of a stimulation condition. To address the issues that come alone with samples containing multiple cell populations with differing scatter properties, draw barcode gates on samples displayed as scatter versus barcode-dye-#1 and scatter versus barcode-dye-#2 (instead of plotting barcode-dye-#1 versus barcode-dye-#2 and drawing one gate). Then use the Populations Manager to create a new population defined by the combination of these two gates. If this approach still doesn't provide enough resolution, you can first gate on cell type using lineage markers before gating on the barcoded populations. While you can barcode based on patient sample, we still recommend barcoding based on stimulation conditions when possible - you lose less information if your barcoding fails, as it would still be possible to use scatter gates to stratify populations and discern which cells types (lymphocytes versus monocytes, for example) had which response.

Q: Why do you export barcoded populations into a new experiment where each population becomes a separate FCS file?

A: By splitting up the files, analysis is much faster in Cytobank as well as other flow cytometry analysis programs. Cytobank gives you convenient tools to automatically export defined barcoded populations into a new experiment where each population defined by barcodes is converted into a separate FCS file. Files with over a million events tend to compute more slowly in all analysis programs, and splitting off your barcode-defined populations into separate FCS files will speed analysis.

Q: Can I analyze barcoded data in another program?

A: Yes, but it will be easier once split into separate files. Cytobank allows you to easily do this.

Q: Which channels are best to use as the barcode channels?

A: A good rule of thumb is to select your barcode channels such that they are on one laser so that they're less likely to bleed into the other channels that you use to gather sample data. This way, the barcode dyes don't need to be compensated out of channels used for quantitative measurements. Some of our resident scientists like using Pacific Orange and Pacific Blue as their barcoding channels, and tend to avoid Alexa 750 because of compensation issues with Alexa 647.

Q: How do barcoding dyes bind to cells?

A: The dyes used are succinimydylester dyes, which bind free amines on proteins of cells.

Q: How can I ensure consistent, reproducible results across multiple experiments?

A: Pre-mix dyes and test them in advance to know they will work at the spacing you have picked. Then, make a batch, pre-aliquot it, and freeze it down at -80*C.

What if my cells divide differentially during the essay? Won't the barcoding dye be diluted, artefactually enriching Dye lo cells?

  --Gilles (Not signed in).....Mon Sep 26 03:42:35 -0700 2011

The barcoding step takes place after cells have been fixed. Therefore, the dye will not be diluted.

  --Angela Landrigan.....Wed Dec 14 18:26:17 -0800 2011