Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry

Background: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. Aim: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)- 5(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. Methods: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn™ ADP Flow Cytometer. Results: The pHi clamping conditions were optimized as 140 mM potassium and 10 μM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂·2H₂O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl₂, 2 mM CaCl ₂·2H₂O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. Conclusion: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.