Petrovic, Katarina, Pfeifer, Martin, Parker, Christian, Tallarico, John, Movva, Rao, Scheel, Guenther and Helliwell, Stephen (2017) Cell viability assays for Saccharomyces cerevisiae: Comparison of fluorescence (Alamar TM Blue) and optical density assays in High Throughput Screening format. Microbiological research., 199. pp. 10-18. ISSN 1618-0623; 0944-5013

Abstract

The budding yeast S.cerevisiae is widely used as eukaryotic model organism in elucidation
of cell physiological pathways and the mechanism of drug action. Its low sensitivity to mechanic effects makes it a good tool for identifying potential drug candidates in high-throughput screening assays conditions. This report describes the development of a set of high throughput methods based on cell viability by monitoring the reduction of Alamar blue (Resazurin) and direct optical measurement of cell growth in 1536-well format. Both methods can be performed using S. cerevisiae in 96, 384 and 1536-well format. Approximately 20’000 compounds from a large pharmaceutical library (~1 million compounds) were identified as active by Almar Blue (Resazurin). From the selected subset, 1’930 concentration response curves were tested in order to access the potency (IC50 values) in a concentration dependent fashion in both assays. This report also describes influences of natural variability of the assays and prolonged plate storage with pre-dispensed compounds on the results of the screen and compares the sensitivity of these two assays in 1536-well plate format.
Summary: We have developed a robust assay to screen for compounds that will inhibit the growth of diploid S. cerevisiae lacking the multi-drug resistance pump gene pdr5. The assay achieves Z’ values of > 0.7 with High to Low ratios of > 6.5 under on-line screening conditions, and makes use of a miniaturized 1536 well format. The screening of yeast by optical density and fluorescence measurement deemed acceptable for high throughput format. Following cell proliferation by measuring optical density seems to be more sensitive as compared to the fluorescence method in determining IC50 values in 1536-well format.

Item Type:	Article
Date Deposited:	15 Apr 2020 00:45
Last Modified:	15 Apr 2020 00:45
URI:	https://oak.novartis.com/id/eprint/26324