A flow injection renewable surface technique for cell-based drug discovery functional assays.
A novel flow injection-renewable surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays on living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface with which the assay is performed. The feasibility of this FI-RS technique is demonstrated by performing a functional assay using Chinese hamster ovary cells transfected with the rat muscarinic receptor (M1). The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calcium-sensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) delivered to cells attached to microcarrier beads. The RS methodology eliminates problems associated with diminishing biological response vis-à-vis traditional functional assays that are performed repetitively on the same group of cells. Using this technique, reproducible responses are measured and pharmacologic parameters quantified that compare favorably to literature values. In addition, the use of the FI-RS functional assay as an analytical method for discrimination of agonists based on kinetic parameters is proposed.
Hodder
P S
PS
Department of Chemistry, University of Washington, Seattle 98195-1700, USA. phodder@u.washington.edu
Ruzicka
J
J
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017022:Flow Injection Analysis / Q000379:methods*; D005456:Fluorescent Dyes; D007700:Kinetics; D018721:Muscarinic Agonists / Q000032:analysis*; D051381:Rats
D005456:Fluorescent Dyes; D018721:Muscarinic Agonists