Measurement of cellular stimulation through monitoring pH changes by bead injection fluorescence microscopy

This work presents a method for extracellular and intracellular pH measurem ents in live cells based on a combination of the bead injection (BI) techni que and fluorescence microscopy. For extracellular pH measurement, cells ar e grown on fluorescent beads, packed into a small column by a sequential in jection instrument, and fluorescence intensity from the beads stained by th e indicator is recorded by a fluorescence microscope. The method is applied to quantifying carbachol stimulation of Chinese hamster ovary (CHO) cells transfected with the mi muscarinic receptor and is verified by a glucose de pletion experiment. The results yield an EC50 value of 1 mu M for carbachol , which is in reasonable agreement with the literature value 3 mu M determi ned by an existing potentiometric technique for measuring acid release. The intracellular measurement utilizes CHO M1 cells growing on non-fluorescent beads. For this method the cells rather than the beads are stained by incu bating them in a Solution of the fluorescent pH indicator BCECF. The cells are also stimulated with carbachol and the intracellular pH dependent fluor escence from the cells is recorded. The results show dependence between int racellular pH changes and carbachol concentration and yield an EC50 value o f 4 mu M. (C) 2000 Elsevier Science B.V. All rights reserved.