Ratiometric measurement of intracellular pH of cultured cells with BCECF in a fluorescence multi-well plate reader |
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Authors: | Roberta L Grant Daniel Acosta |
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Institution: | (1) Division of Pharmacology & Toxicology, College of Pharmacy, The University of Texas at Austin, 78712-1074 Austin, Texas |
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Abstract: | Summary 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 μM BCECF for 30 min at 37° 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 ionophore.
Low doses of ionomycin (2.5–5 μM), produced a prolonged acidification; 7.5 μM ionomycin produced a transient acidification; and 10 μM 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. |
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Keywords: | corneal epithelial cells pH primary cultures toxicity screening cytotoxicity |
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