Barcoding - Text-based tutorial

Background

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 [1,2]. 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 use barcoding to distinguish stimulation conditions; however, barcoding can be applied to any distinct populations, such as patient samples or different time points of a stimulation condition.

In the Dataset #8938, human PBMCs were stimulated with four different stimuli, and two unstimulated conditions were included, resulting in the need to barcode such that these six sample types can be distinguished. For this experiment, two dyes were used for barcoding – Pacific Orange (PacO) having three staining intervals (L0, L1, L2), and Alexa 750 (Ax750) with two staining intervals (L0, L1), resulting in a 3 by 2 barcoding matrix. Using Cytobank, you can computationally separate 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.

How To Analyze these Data On Cytobank

  1. Open Dataset #8938 and clone it.

  2. Enter the Gating Interface. Gate on Intact cells (plotting FSC-A versus SSC-A), then singlets (plotting FSC-A versus FSC-W).

  3. Next, we'll decode the 3 x 2 barcode matrix defined by differing levels of PacO and Ax750. The best resolution of barcoded populations is achieved by viewing each fluorophore against a scatter channel, and then drawing gates. For example, plot SSC-A versus PacO and draw three gates to define each barcoded population. Then plot SSC-A versus Ax750 and draw two gates to define each of those barcoded populations.

  4. Scroll down the page to the Population Manager. Each of your stimulation conditions of interest is defined by combinations of different intensities of PacO and Ax750, in addition to being children of Intact Cells and Singlets. To define these populations, click the "Create Population" button, and then check the boxes that define that particular stimulation condition.

  5. In this experiment, create the populations (based on the stimulation conditions) as follows: the first unstimulated sample is defined by Ax750-L0 and PacO-L0; the IFN-treated sample is defined by Ax750-L0 and PacO-L1; the PMA-treated sample is defined by Ax750-L1 and PacO-L0; the LPS-treated sample is defined by Ax750-L1 and PacO-L1; and the second unstimulated sample is defined by Ax750-L1 and PacO-L2. (Also check the boxes for "Intact cells" and "Singlets" for all of these.) Notice that the first unstimulated sample contains the lowest concentration of both dyes, while the second unstimulated sample contains the highest concentration of both dyes. High level of barcoding dye can sometimes alter antibody staining, so both of these unstimulated controls are included for reference.

  6. Return to the Working Illustration. A common way to proceed with analysis is to "deconvolute" or "debarcode" the data by converting each barcoded population into its own FCS file for further analysis. To do this, we'll make an illustration that shows all of the populations, and then export this to a new experiment. First select each of the populations within the Populations Figure Dimension box, making sure to uncheck the Ungated box. Select one channel from each of the two staining panels. Cytobank will automatically bring over data for all of the channels associated with those two panels when you make the clone. Recall that our barcode-only stain is assigned to Panel 3, and we won't be using Panel 3 data today.

  7. In the Experiment navigation bar, click to Actions > Cloning > Split Files by Population. This will create a new experiment with 12 FCS files – six FCS files representing each of the gated populations, for each of two staining panels. Each file is assigned the name of the stimulation condition in the suffix. From here, analysis proceeds as usual for the newly created experiment. Because these files are derived from sub gates of Intact Cells and Singlets, you won't have to draw those gates on these files, and can proceed directly to drawing gates to define populations of interest.

 

References:

[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.



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