RATIOMETRIC MEASUREMENT OF INTRACELLULAR PH OF CULTURED-CELLS WITH BCECF IN A FLUORESCENCE MULTIWELL PLATE READER

A number of methods have been developed to measure intracellular pH (p Hi) because of its importance in intracellular events. A major advance in accurate pHi measurement was the development of the ratiometric fl uorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescei n (BCECF). We have used a fluorescence multi-well plate reader and a r atiometric method for determining pHi in primary cultures of rabbit co rneal epithelial (CE) cells with BCECF. Fluorescence was measured at e xcitation wavelengths of 485 +/- 11 nm and 395 +/- 12.5 nm, with emiss ion detected at 530 +/- 15 nm. Cells grown in multi-well plates were l oaded with 4 mu M BCECF for 30 min at 37 degrees C. Resting pHi was 7. 34 +/- 0.03 (2 cultures, N = 5 wells). Changes in pHi determined with the fluorescence multi-well plate reader after the addition and remova l of NH4Cl or sodium lactate were comparable to changes in cells analy zed with a digitized fluorescence imaging system. A concentration-resp onse relationship involving changes in pHi was easily demonstrated in CE cells after treatment with ionomycin, a calcium ionophore. Low dose s of ionomycin (2.5-5 mu M), produced a prolonged acidification; 7.5 m u M ionomycin produced a transient acidification; and 10 mu M ionomyci n resulted in a slight alkalinization. We conclude that accurate pHi m easurements can be obtained with a ratiometric method with BCECF in a multi-well plate reader. This technology may simplify screening studie s evaluating effects of hormones, growth factors, or toxicants on pHi homeostasis.