Ratiometric measurement of intracellular pH of cultured cells with BCECF in a fluorescence multi-well plate reader.

A number of methods have been developed to measure intracellular pH (pHi) because of its importance in intracellular events. A major advance in accurate pHi measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We have used a fluorescence multi-well plate reader and a ratiometric method for determining pHi in primary cultures of rabbit corneal epithelial (CE) cells with BCECF. Fluorescence was measured at excitation wavelengths of 485 +/- 11 nm and 395 +/- 12.5 nm, with emission detected at 530 +/- 15 nm. Cells grown in multi-well plates were loaded with 4 microM 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 removal of NH4Cl or sodium lactate were comparable to changes in cells analyzed with a digitized fluorescence imaging system. A concentration-response relationship involving changes in pHi was easily demonstrated in CE cells after treatment with ionomycin, a calcium ionopore. Low doses of ionomycin (2.5-5 microM), produced a prolonged acidification; 7.5 microM ionomycin produced a transient acidification; and 10 microM ionomycin resulted in a slight alkalinization. We conclude that accurate pHi measurements can be obtained with a ratiometric method with BCECF in a multi-well plate reader. This technology may simplify screening studies evaluating effects of hormones, growth factors, or toxicants on pHi homeostasis.