Automation and Validation of a High-Throughput In Vitro Toxicity Screen using Promega’s
CytoTox-ONE Homogeneous Membrane Integrity Assay and Amphioxus Technologies
ACTIVTox® Human Hepatocyte Cell Line
Tanya Guthrie, Melinda Ingrum, Phillip Bradford, Jennifer Fiedler (Southern Research Institute), Monika Walterscheid (Amphioxus Cell Technologies), Pam Guthmiller (Promega Corporation),
Amy Rizvi, and Thomas M. Fletcher III (Southern Research Institute)
These studies were undertaken to evaluate the use of Promega’s
CytoTox ONE Homogeneous Membrane Integrity Assay in a high
throughput format. This assay was designed to test the toxicity of a
compound by measuring the release of lactate dehydrogenase (LDH) in the
surrounding media that occurs upon membrane degradation. Additionally,
cell viability was determined following the LDH measurements by using a
luminescent ATP concentration assay.
ACTIVITox Human C3A hepatocyte cell line was used as the target cell
line in this study. C3A is a unique cloned hepatic cell line that simulates
normal human liver responses to stimuli, including drugs and stress. The
validation and optimization results performed on a Beckman Coulter Core
Rail System will be presented here. The studies were performed with the
support of the NIH-NIAID Tuberculosis Drug Screening Contract (N01-AI-
15449) for use in Mycobacterium tuberculosis primary screen compound in
vitro toxicological evaluations.
•Special thanks to Pam Guthmiller of Promega Corporation for providing
product graphics and technical assistance.
•Cells were kindly provided by Amphioxus Cell Technologies.
•This work was supported in part by NIH-NIAID contract NO1-A1-15449,
Tuberculosis Drug Screening
•Fast: The plates are incubated for 10 minutes before reading, compared to 30
minutes or more with classic LDH assays. This increases output allowing the
capability to screen large compound libraries
•Automatable:. The validation data provided a Z-factor value that ensure