Fluorescent cell barcoding for multiplex flow cytometry

Abstract

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.

Figure 6.31.1

Fluorescent Cell Barcoding protocol. Cell samples are labeled with an amine-reactive fluorescent dye (FCB dye) at different concentrations. After covalent labeling has occurred, cells are washed to remove unbound dye, then combined into one tube and stained with antibodies against intracellular or surface antigens. The combined tube is then acquired on the cytometer. After acquisition, the original cell samples are identified by gating populations that display discrete fluorescent intensities in the FCB channel.

Figure 6.31.2

Deconvolution of four barcoded samples. Four independent tubes of U937 cells were barcoded with 0, 0.016, 0.063, or 0.25 µg/ml AlexaFluor 488 (Ax488), then combined into one tube and run on the flow cytometer. After identifying singlet events, intact cells are gated based on forward and side scatter characteristics. Plotting the FCB channel (Ax488 in this case) versus side scatter reveals four distinct populations that correspond to the four original tubes that were barcoded. Once gated, the populations can be analyzed for other antigens of interest.

Figure 6.31.3

Layout of barcoding matrix used to encode 27 samples. Three FCB dyes were used: DyLight 350, Pacific Orange, and DyLight 800. Each dye was used at three concentrations. DyLight 350 encoded the three rows while the combination of Pacific Orange and DyLight 800 encoded the nine columns.

Figure 6.31.4

Deconvolution of 27 barcoded primary cell populations. 27 individual wells were barcoded using all the unique combinations of DyLight 350 at 0, 0.5, or 2 µg/ml; Pacific Orange at 0, 0.25, or 1 µg/ml, and DyLight 800 at 0, 0.25, or 1 µg/ml. Cell events were first identified by gating on singlets (FSC-area vs. -width) and cells (FSC-area vs. SSC-area) as in Figure 6.31.2. Three populations are then clearly visible when plotting any of the barcoding parameters against side scatter. In this analysis, the rows were first gated based on staining intensity in the DyLight 350 parameter. The three rows were then analyzed for Pacific Orange staining, which was used to separate the columns into groups of three. Analyzing each of the levels of Pacific Orange for DyLight 800 revealed the samples from individual wells C1–C9. This analysis was repeated for each row, yielding 27 gated populations. The gating of the individual wells within rows A and B are not shown.