Multiplexed labeling of viable cells for high‐throughput analysis of glycine receptor function using flow cytometry

Abstract

Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy. ' 2009 International Society for Advancement of Cytometry Key terms multiplex; chloride channels; glycine receptor FLOW cytometry permits multiple cell parameters to be analyzed simultaneously with high sensitivity and a high degree of statistical robustness. It is emerging as an important drug discovery tool because it can screen test compounds against a wide variety of cellular targets simultaneously. However, achieving this goal is dependant on two factors: (1) the ability to automate the screening of large numbers of compounds, and (2) the ability to monitor multiple parameters simultaneously in live cells. Recent developments (reviewed by Sklar et al. (1)), while useful, require dedicated hardware on an unmodified flow cytometer.Multiplexed or duplexed analysis allows the screening of multiple discrete samples in one tube or well at the same time (2-4). A recent method (5), which introduced the idea of labeling discrete cell populations with a unique combination of fluorochromes, demonstrated the feasibility of employing flow cytometry to discriminate between different multiplexed labeled cell populations. However, this method required cellular fixation, preventing further culture or experimentation.Glycine receptors (GlyRs) are pentameric Cys-loop anion-permeant channels that mediate inhibitory neurotransmission in the spinal cord, brainstem, and retina (6,7). These receptors have recently emerged as therapeutic targets for chronic inflammatory pain and movement disorders (8-10). Our interest lies in discovering novel bioactive compounds with therapeutic potential and i...

Fluorescent target array killing assay: A multiplex cytotoxic T‐cell assay to measure detailed T‐cell antigen specificity and avidity in vivo

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Pathophysiological Mechanisms of Dominant and Recessive GLRA1 Mutations in Hyperekplexia

“…Cells were imaged using an automated high-content screening system using YFP-I152L fluorescence as an indicator of Cl Ϫ influx rate (Kruger et al, 2005;Gilbert et al, 2009). Briefly, HEK293 cells were cotransfected with mutant or WT pRK5-hGlyR␣1 and pcDNA3.1-YFP-I152L and plated into a 384-well plate (ϳ2.5 ϫ 10 3 cells/well).…”

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses <em>In vivo</em>

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