Rapid comparative immunophenotyping of human mesenchymal stromal cells by a modified fluorescent cell barcoding flow cytometric assay

Abstract

Flow cytometry immunophenotyping is a sensitive technique allowing rapid characterization of single cells within heterogeneous populations, but it includes several subjective steps during sample analysis that impact the development of standardized methodology. Proposed strategies to overcome these limitations include fluorescent cell barcoding (FCB), which facilitates multiplexed sample evaluation with increased data reproducibility whilst reducing labeling variation, materials, and time. To date, the FCB assay has found utility for analyzing the phosphorylation status of intracellular targets but has not been intensively employed for cellular immunophenotypic analyses using cell surface markers. In this study we developed a modified FCB assay for multiplexed analysis of human mesenchymal stromal cells (hMSCs) to evaluate the quality of these cells during bioprocessing. A panel of fluorochrome-conjugated antibodies was used to target 15 ubiquitously expressed or stage-specific markers together with a fixable viability dye eFluor 506 acting as the cell barcoding agent. Critical technical considerations and validation steps were presented in the context of monitoring hMSC status, defined by generic, and specific surface markers for cell identity and quality. It was found that at discrete passages, inter-analyst expression patterns between hMSCs cultures were similar, but in contrast, diverse marker expression was evident between passages. A side-by-side analysis of barcoded and non-barcoded cells demonstrated the potential of this technique for the rapid phenotypic characterization of cells exposed to different bioprocessing conditions. Additionally, the method incorporates fewer subjective factors; including sample preparation and instrument day-to-day variations and is customizable across a diversity of cell types. © 2017 International Society for Advancement of Cytometry.

High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis

“…FCB is a multiplexed high-throughput flow cytometry technique which has been used for phosphoFlow analysis [1][2][3][4][5][6], detection of intracellular cytokines [7], and immunophenotyping of human PBMCs and mesenchymal stem cells [9][10]. In recent years, mass cytometry has gained popularity for phosphoprotein profiling, and has been frequently used for studies aimed at quantifying relative changes.…”

“…Fluorescent cell barcoding (FCB), a high-throughput flow cytometry (FCM) technique for multiplexed assays, was first developed for single-cell phospho-specific FCM (phosphoFlow) [1][2][3][4][5][6], and subsequently optimized for detection of intracellular cytokines [7][8] and immunophenotyping [1][2][3][4][5][6][7][8][9][10]. FCB minimizes staining variability and decreases both antibody consumption and required sample volumes [1,11].…”

“…The advantage in performing large-scale studies by the FCB technique is to generate more reliable FCM data by minimizing technical variability, as all samples and controls are processed and acquired collectively [1]. For these reasons, FCB is particularly applicable to translational studies, and new barcoding methods are being developed using amine-reactive or fixable viability dyes [9][10]12].…”

“…Standardization of protocols and antibody panels in FCM for phenotyping is important for more reliable, comparable, and reproducible data, and, especially critical for samples collected during clinical trials [28][29][30][31]. The use of FCB minimizes inter-assay variations, as all samples are acquired together, but a rigorous standardization and optimization of the protocol is still required before implementation of the technique both in clinical and basic research settings [1][2][3][4][5][6][7][8][9][10]. However, analysis of FCB data is usually conducted by conventional manual gating strategies using available commercial software products, which are designed to analyze individual samples by a gating sequence limited to one or two parameters at a time.…”

Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry

“…Such strategy can be used to analyze samples under treatments, co-cultured, or stimulated with different agents. FCB has been successfully utilized for different studies, including drug screening and cell signaling profiling studies [26], intracellular cytokine production [27], immunophenotyping [28], and others.…”