Microplate live cell assay system for early events in mechanotransduction.
For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.
Graff
Ronald D
RD
Department of Orthopaedics and Thurston Arthritis Research Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. rgraff@med.unc.edu
Lee
Greta M
GM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002452:Cell Count; D002470:Cell Survival; D002478:Cells, Cultured; D049454:Firefly Luciferin; D008156:Luciferases; D008163:Luminescent Measurements; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008832:Microchemistry; D013314:Stress, Mechanical; D013552:Swine
D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D008156:Luciferases; D002118:Calcium