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Application of a Multiplexed High Content Imaging (HCI) Based Cell Viability and Apoptosis Chemical Screening Assay with Results in MCF-7 Cells
Citation:
Willis, C. AND J. Harrill. Application of a Multiplexed High Content Imaging (HCI) Based Cell Viability and Apoptosis Chemical Screening Assay with Results in MCF-7 Cells. Society of Toxicology, San Antonio,TX, March 11 - 15, 2018.
Impact/Purpose:
This study demonstrated the multiplexed assay’s ability to effectively detect chemicals which produce changes in either cell viability or apoptosis. Future directions include applying this assay to additional cell lines and examining the relationship between nucleus morphology (i.e. size and texture) and decreases in cell viability or increases in apoptosis. Presentation at Society of Toxicology meeting March 2018.
Description:
The NCCT high throughput transcriptomics (HTTr) screening program uses whole transcriptome profiling assay in human-derived cells to collect concentration-response data for large numbers (100s-1000s) of environmental chemicals. To contextualize HTTr data, chemical effects on cellular function were examined using a high content imaging-based, multiplexed cell viability and apoptosis assay based on propidium iodide (PI) dye exclusion and caspase 3/7 activation. MCF-7 cells were plated in a 384-well format (10,000 cells/well) utilizing a BioTek MultiFlo FX dispenser. Following a 24h recovery period, a total of 1,804 chemicals were tested at 8 concentrations (semi-log spacing up to 100 µM) for a single exposure duration (6 hr). A positive control chemical (ionomycin) was applied in duplicate to each plate to evaluate assay performance. Chemicals were applied to each assay plate using an ECHO 550 acoustic dispenser in a randomized pattern unique to each assay plate. After treatment, cells were live-stained with Hoechst 33342 (nuclei), PI (cell viability), and CellEvent Caspase-3/7 Green Detection Reagent (apoptosis) for 30 minutes before being fixed and imaged using an Opera Phenix high content screening system. A workflow was developed using Harmony cellular imaging software. The resulting cell-level measurements of PI and activated caspase 3/7 fluorescent intensity were exported and analyzed utilizing customized scripts in R. The median Z-factor across plates for cell viability and apoptosis endpoints was 0.70 and 0.50, respectively. Significant concentration-dependent decreases in cell viability or increases in apoptosis exceeding a threshold of 15% were observed for 65 and 42 chemicals respectively. Of these chemicals, 32 were active for both endpoints. In a follow-up study, increasing exposure duration resulted a greater percentage of active chemicals. In conclusion, this study demonstrated the multiplexed assay’s ability to effectively detect chemicals which produce changes in either cell viability or apoptosis. Future directions include applying this assay to additional cell lines and examining the relationship between nucleus morphology (i.e. size and texture) and decreases in cell viability or increases in apoptosis. This abstract does not necessarily reflect USEPA policy.
URLs/Downloads:
WILLIS.SOT.2018.APOPCV.ABSTRACT_FINAL.PDF (PDF, NA pp, 81.651 KB, about PDF)SOT 2018 APOPCV POSTER WILLIS_V2.PDF (PDF, NA pp, 703.044 KB, about PDF)