Raw data for TMA-DPH decay-fluorescence in Prola 2020-SciAdv

1-(4-Trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA‑DPH, Sigma Aldrich) was used to monitor physical properties of mitochondrial membranes of 7-to-8-mo-old mice. TMA‑DPH is composed of a cationic substitute (TMA) that anchors at the polar heads of the membrane, while allowing the fluorescent hydrophobic probe DPH to be located in nonpolar regions. To limit fluorescent noise, mitoplasts were resuspended in the Hypotonic Buffer (sucrose 9 mM, mannitol 29 mM, Hepes 0.3 mM, pH 7.4) as described hereafter.

Experiments were repeated five times using independent samples, each from different mice. Decay-fluorescence measurements were performed on each sample at 37 °C after a measurement of the instrumental response of the spectrofluorimeter used to measure fluorescence-decay (“prompt”). Thirty µl of sample (DO₆₀₀ = 0.055) diluted in 2.97 ml of Hypotonic Buffer were introduced into spectroscopic quartz cuvette with an optical path length of one cm (VWR International).

Fluorescence-decay was measured by the time-correlated single-photon counting (TCSPC) method using a Horiba-Fluoromax-4® spectrofluorimeter (Horiba) equipped with a 370-nm laser diode (NanoLED C2, Horiba) as the source of excitation. Fluorescence decays were measured in TCSPC setup (Deltahub, Horiba). The Instrument Response Function (IRF) was about 160 ps (measured at 370 nm using the hypotonic buffer). Emission and excitation wavelength of TMA-DPH were respectively fixed at 370 nm and 431 ± 1.1 nm. Each decay curve corresponded to 10,000 counts.