Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling

Abstract

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

“…Fluorescent cell barcoding (FCB) represents another, flow cytometry-based labeling technique, which utilizes fluorescent dyes for staining of fixed cells resulting in a unique signature due to defined fluorescence intensities and emission wavelengths for different samples. 13 However, tracking of individual clones cannot be achieved in vivo and the mix can only be quantified at single time points. 13 A recent advancement of the FCB approach, termed fluorescent genetic barcoding (FGB), harnesses the retroviral expression of up to three fluorescent proteins additionally stratified by two different fluorescence intensities.…”

“…13 However, tracking of individual clones cannot be achieved in vivo and the mix can only be quantified at single time points. 13 A recent advancement of the FCB approach, termed fluorescent genetic barcoding (FGB), harnesses the retroviral expression of up to three fluorescent proteins additionally stratified by two different fluorescence intensities. 14 The FGB technique permits clonal cell behavior monitoring over time in vitro for up to 12 different clones, as long as fluorescence signals of analyzed cells are bright enough to facilitate clear-cut identification of two stacked intensity levels.…”

A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking

“…As a consequence, the 1G-FGB system appears most suitable for applications that permit the enrichment of pure color-coded populations for longitudinal multiplexing studies, while circumventing the need for FCB-mediated staining procedures. 14,15 To accelerate the generation of double and triple marker expressing color-coded cells, we next developed a silencing-resistant coexpression system based on a chimeric CBX3-SFFV promoter and 2A cleavage sites (Figure 2A). Owing to cargo size constrains of the lentiviral vector system, 27 we utilized a minimal 700 bp CBX3 fragment with proven anti-silencing function in P19 and induced pluripotent stem cells.…”

“…14 In this context, the lentiviral backbone supports gene transfer into a variety of cell types including populations that are traditionally difficult to transduce, such as hematopoietic stem cells (HSCs), neurons, and liver cells. 7,19,20 As genetically encoded fluorescent proteins, we chose GFP, YFP, and meKO2 (a modified form of the monomeric KusabiraOrange; Figure S1), because of their sharp fluorescent profiles and their compatibility with immunophenotyping applications (e.g., APC, APC-Cy7, PE-Cy7, AF700, eVolve605, DAPI/eF450/Pacific Blue, and PI) in multicolor flow cytometry.…”

“…13 Therefore, multiplex flow cytometry assays have been developed on the basis of "fluorescent cell barcoding" (FCB). 14 This technique relies on the staining of individual samples with a unique color code prior to sample pooling and optional epitope staining before simultaneously acquiring all samples from a single tube. Since the number of color codes increases geometrically through multiplexing the number of dye concentrations with the number of fluorophores; e.g., two different concentrations of three dyes yield 27 color codes, FCB offers the potential for reducing reagents and increasing comparability of staining results.…”