title: Reproducibility and robustness of a real-time microfluidic cell toxicity assay.
abstract: Numerous opportunities exist to apply microfluidic technology to high-throughput and high-content cell-based assays. However, maximizing the value of microfluidic assays for applications such as drug discovery, screening, or toxicity evaluation will require assurance of within-device repeatability, day-to-day reproducibility, and robustness to variations in conditions that might occur from laboratory to laboratory. This report describes a study of the performance and variability of a cell-based toxicity assay in microfluidic devices made of poly(dimethylsiloxane) (PDMS). The assay involves expression of destabilized green fluorescent protein (GFP) as a reporter of intracellular protein synthesis and degradation. Reduction in cellular GFP due to inhibition of ribosome activity by cycloheximide (CHX) was quantified with real-time quantitative fluorescence imaging. Assay repeatability was measured within a 64-chamber microfluidic device. Assay performance across a range of cell loading densities within a single device was assessed, as was replication of measurements in microfluidic devices prepared on different days. Assay robustness was tested using different fluorescence illumination sources and reservoir-to-device tubing choices. Both microfluidic and larger scale assay conditions showed comparable GFP decay rates upon CHX exposure, but the microfluidic data provided the higher level of confidence.
authors.affiliation: Biochemical Science Division, NIST, Gaithersburg, Maryland, USA. gregory.cooksey@nist.gov
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Cooksey
authors.forename: John T
authors.initials: JT
authors.lastname: Elliott
authors.forename: Anne L
authors.initials: AL
authors.lastname: Plant
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000737:chemistry / Q000633:toxicity; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*; D014709:Vero Cells
chemical_list: D004129:Dimethylpolysiloxanes; D049452:Green Fluorescent Proteins; C013830:baysilon; D003513:Cycloheximide