Intracellular pH Measurement in HEK 293 Cells

HEK 293 cells were loaded with SNARF-5F-acetoxymethyl ester (Life Technologies Corporation) for 30 min at 37 °C. The same perfusion system in Ca²⁺ imaging was used to apply acid solutions. SNARF dye was excited with 488 nm laser in NIS elements (Nikon), and the ratio of fluorescence emissions at 640 and 580 nm (F640/F580) were captured every 5 s using a Nikon C2Si camera and Image Suite software (Nikon). Intracellular pH were measured by calibration curve obtained from modified HBSS at different pH30 (link)31 (link).

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Sanematsu K., Kitagawa M., Yoshida R., Nirasawa S., Shigemura N, & Ninomiya Y. (2016). Intracellular acidification is required for full activation of the sweet taste receptor by miraculin. Scientific Reports, 6, 22807.

Publication 2016

NIS elements C2Si camera

Acid Ester Fluorescence Hbss Hek 293 cells Intracellular Snarf dye

Corresponding Organization :

Other organizations : Kyushu University, Japan International Research Center for Agricultural Sciences

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Variable analysis

independent variables

Application of acid solutions

dependent variables

Intracellular pH measured by SNARF-5F-acetoxymethyl ester fluorescence ratio (F640/F580)

control variables

HEK 293 cells
SNARF-5F-acetoxymethyl ester loading
Perfusion system used in Ca2+ imaging
SNARF dye excitation at 488 nm
Fluorescence emission ratio at 640 and 580 nm
Calibration curve obtained from modified HBSS at different pH

controls

Modified HBSS at different pH values used as a positive control for the calibration curve

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