Cellular pH Measurement with Firefly Luciferase

Native Fluc spectra were collected in solution using a Cary Eclipse Fluorescence Spectrophotometer (Agilent) with 10 nM Quantilum (Promega, Madison, WI; E1701) in 30 mM MOPS, 10 mM β-mercaptoethanol, 1 mM ATP pH 7.4, 1 mM potassium luciferin, 10 mM MgSO₄, and 1 mg/mL bovine serum albumin, as described previously^{101 (link)}. Cellular pH calibration was performed with stably transfected SV40:Luc U2OS cells at 37 °C in white 96 well plates (Corning™ 3917) on a Tecan Spark 10 M, alternately using 550–575 nm and 610–635 nm filters to calculate 560/620 nm ratio (Supplementary Fig. 2c, d) with 5 s integration times. At the depicted timepoint, 10 μL of 10 μM FCCP was automatically injected to give a final concentration of 1 μM FCCP. Longitudinal measurements were also performed on a Tecan Spark 10 M (Supplementary Fig. 2c, d), under the same conditions, measuring total bioluminescence from each well (1 s integration) in addition to emission at 550–575 and 610–635 nm from the same well (5 s integration). For these experiments cell media was buffered with 20 mM MOPS instead of bicarbonate, as described previously⁸⁸ . The pH of the various media was measured and adjusted at 37 °C with 1 M HCl or NaOH, and then adjusted to 342 mOsm with 1 M NaCl.