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.


OBJECTIVE
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 microM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).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.


CONCLUSIONS
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.
Koo
Mei K
MK
Department of Physiology, Yong Loo Lin School of Medicine, National University Medical Institutes, Singapore.
Oh
Chia H
CH
Holme
Andrea L
AL
Pervaiz
Shazib
S
D001578:Benzopyrans; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism*; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry; D007476:Ionophores / Q000494:pharmacology; D047569:Laser Scanning Cytometry / Q000379:methods*; D009550:Nigericin / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism
D001578:Benzopyrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007476:Ionophores; C063917:SNARF dye; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D009550:Nigericin; D011188:Potassium