Abstract
Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.
Highlights
Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis
To determine, which cells were initially loaded after the cut with a steel blade, and verify, that a dye transfer occurs through intercellular gap junctions, the traditional scrape loading-dye transfer (SL-DT) uses another membrane impermeable fluorescent dye, such as Texas red-dextran (MW 10,000), www.nature.com/scientificreports concurrently with the gap junction diffusional dye
The “#” denotes significant differences at P ≤ 0.05 compared to the vehicle control (Kruskal-Wallis ANOVA with Dunnett’s test). (c) The representative images of communicating cells stained with Lucifer Yellow (LY), the initial loaded cells along the cut stained with propidium iodide (PI), the total cells stained with Hoechst 33342 (HB+) and dead cells stained with PI (PI+)
Summary
Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. Currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. In vitro assessment of GJIC has been used relatively less frequently in the modern research in comparison to other and more traditional phenotypic assays, such as evaluation of cellular metabolic activity, cell proliferation, cell cycle, autophagy, apoptosis, cell migration, cytoskeletal rearangements[13] This might be partially due to the until-recent lack of simple, rapid, accessible and affordable methods allowing in vitro GJIC assessment in a higher-throughput workflow. In contrast to the traditional assay, this innovative multiparametric version of SL-DT, coupled with (semi)-automated image acquisition and analysis for all of the evaluated parameters, allows to increase the throughput of this assay and provide much faster and less expensive approach for GJIC assessment compatible with in vitro high-content analysis/screening (HCA/HCS) of pharmacologically and toxicologically significant compounds
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