Multiplexing Fluo-4 NW and a GeneBLAzer transcriptional assay for high-throughput screening of G-protein-coupled receptors.
Activation of G-protein-coupled receptors (GPCRs) leads to a cascade of signaling events, including calcium mobilization and downstream transcriptional activation of various proteins. Two commonly used methods of high-throughput screening for GPCRs include calcium-sensitive dyes, such as Fluo-4 NW, and reporter gene assays, such as beta-lactamase. To determine whether the advantages of each assay format could be combined by multiplexing, Jurkat and CHO-K1 cell lines over-expressing the M1 muscarinic receptor and beta-lactamase under control of an NFAT response element were tested in a multiplexed format. The Jurkat cell line was further screened with a subset of the LOPAC(1280) library. The multiplexing assay was compatible with both the CHO-K1 and Jurkat cell lines. For the screen, there was 100% correlation of on-target hits in the multiplexed format, and several false positives with each assay format were identified. Therefore, not only can the assays be multiplexed, but by multiplexing, the false positives associated with each assay format also could be easily identified. In addition to enhanced reliability, this method saves time and money because only half the amount of compounds, cells, and consumables are needed to screen a cell line in a multiplexed mode versus separate screening by both methods.
Hanson
Bonnie J
BJ
Invitrogen Corporation, Madison, Wisconsin 53719, USA. bonnie.hanson@invitrogen.com
D000814:Aniline Compounds / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay / Q000379:methods; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014966:Xanthenes / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D000814:Aniline Compounds; C409648:Fluo 4; D043562:Receptors, G-Protein-Coupled; D014157:Transcription Factors; D014966:Xanthenes; D001618:beta-Lactamases