Application of division arrest technology to cell-based HTS: comparison with frozen and fresh cells.
Cell-based functional assays are becoming popular in many HTS laboratories because of recent advances in detection and automation technologies. However, the supply of large amounts of live cells with consistent cellular response for day-to-day screening operations over several days/weeks is a tremendous challenge. The high cost of cell culture, labor-intensive nature of the work, and inherent variability in cellular responses from time to time tend to be prohibitive for extensive applications of cell-based assays in HTS. We therefore tested division-arrested cells that were prepared in a single batch and frozen at -80 degrees C before use in several cell-based assays and in a robotic screening campaign. Chinese hamster ovary cells expressing a Gq-coupled receptor were analyzed for the agonist-induced intracellular Ca2+ response measured on a fluorescent imaging plate reader. In this case, the division-arrested cells showed consistent agonist-induced intracellular Ca2+ concentration response as reflected by signal-to-basal ratio and EC50 even 48 h after cell plating. In comparison, the responses from untreated frozen cells and fresh cells declined significantly approximately 30 h after cell plating. In other cell-based assays tested (cyclic AMP assay, reporter gene beta-lactamase assay, and ion-channel assay), the division-arrested cells performed as well as frozen, or fresh cells. We thus conclude that the use of alternate strategies such as frozen cells or division-arrested cells may alleviate the need for several batches of cell plating each day during HTS while maintaining the desired robotic throughput and assay quality.
Kunapuli
Priya
P
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA. priya_kunapuli@merck.com
Zheng
Wei
W
Weber
Michael
M
Solly
Kelli
K
Mull
Rebecca
R
Platchek
Michael
M
Cong
Mei
M
Zhong
Zhong
Z
Strulovici
Berta
B
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002118:Calcium / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015925:Cryopreservation / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D016685:Mitomycin / Q000494:pharmacology*; D015203:Reproducibility of Results; D012371:Robotics / Q000379:methods; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D016685:Mitomycin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium