Category: Automation and High-Throughput Technologies

1247-C - A fully automated long-term cell-based assay identifies compounds with delayed cytotoxicity

Tuesday, February 6, 2018
2:00 PM - 3:00 PM

Many high throughput chemical screens are designed to identify compounds with acute cytotoxicity that show activity within 1 – 3 days.  We were interested in compounds that have a delayed effect, showing cytotoxicity at time points longer than 3 days.  Here, we describe a 384-well, 12-day cytotoxicity assay using a non-adherent Acute Lymphoblastic Leukemia cell line, NALM6 carrying a mutant CREBBP gene, screened in a dose-responsive manner.  These cells proliferate rapidly, doubling approximately every 35 hours, so a 12-day incubation required them to be sub-cultured on day 4 and again on day 8 before reading the assay on day 12.  Each time we sub-cultured the cells, we dosed the child plate with fresh compounds and measured the viability of the parental plate after sub-culturing.  The day 12 viability was measured without sub-culturing prior to reading.  For each of the 222 compounds screened, we measured the cellular response to a 10-point serial dilution series in triplicate.  As expected, we observed an increase in the variability in the assay with each passage; this caused a reduction in our z’-factors over the course of the assay.  For each time point, the data were fitted with dose response curves, and IC50 values were calculated.  We were interested in compounds that showed activity on day 8 or day 12 but no activity on day 4.  To select hits, compounds were filtered to keep only those that showed no activity on day 4, but that did show activity on either day 8 or 12.  To further refine our hit list, we removed compounds with an IC50 value greater than 5 µM and those with a maximal inhibitory activity less than 50%, expressed in our analysis as having an activity greater than -50%.  This filtering strategy resulted in 11 hits that induced the desired delayed activity.  This study was greatly facilitated by the automation we used to sub-culture the cells; our focus here is the development of the robotic procedures that enabled this study.

Duane Currier

Sr HTS Automation Technologist
St Jude Children
Memphis, Tennessee

Duane Currier is a Senior Automation Technologist in the High Throughput Bioscience Center, a division of the Chemical Biology and Therapeutics Department at St. Jude Children's Research Hospital. Working with the other members of his team, he provides complete automation support to the varied high throughput projects that approach the center for assistance.
Duane received his Master’s degree in Biochemistry, Cellular, and Molecular Biology from the Johns Hopkins University School of Medicine. Though trained as a multidisciplinary biologist, Duane has demonstrated a refined talent for laboratory automation and computer science over a five year period at USAMRIID and now St. Jude. While the robots are working, he develops software to enhance the productivity of his team and drive the optimization of key processes within the department.