Flow cytometric measurement of cytoplasmic pH: A critical evaluation of available fluorochromes

Three pH‐sensitive fluorochromes–4‐methyl‐umbelliferone(4MU),2,3‐dicyano‐hydroquinone (DCH), and 2′,7′‐bis(carboxyethyl)‐5,6‐carboxy fluorescein (BCECF)–were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pH i ) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pH i , and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pH i , which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore. Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pH i measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.