Simultaneous measurement of Ca2+ and pH by laser cytometry using fluo-3 and SNARF-1

We present a new convenient method for simultaneous measurement of intracellular calcium concentration ([Ca2+]i) and intracellular pH (pHi) using laser cytometry with a mixture of fluo-3 (for [Ca2+]i) and SNARF-1 (for pHi), with iso excitation (488 nm)-dual emission (530 nm for fluo-3 and > 630 nm for SNARF-1). By using this technique, we measured the changes in [Ca2+]i and pHi in A-431 human epidermoid carcinoma cells and HSG human salivary gland cells stimulated by ATP. We found that alkalization in A-431 cell occurred with the elevation of [Ca2+]i; in contrast, alkalization in HSG cells did not occur at all, even though the elevation of [Ca2+]i was observed. Using BAPTA (a chelating agent of Ca2+) and amiloride (an inhibitor of the Na+/H+ exchanger), we found that the elevation of pHi requires the elevation of [Ca2+]i but that the elevation of [Ca2+]i does not always require a rise in pHi. From our results we conclude that elevation of [Ca2+]i takes precedence over the elevation of pHi in ATP-stimulated signal transduction.