Multiscan sky

Arabidopsis I + III₂ supercomplex purified by sucrose gradient ultracentrifugation was tested for activity, using an established protocol^{59 (link)}. The assay was carried out in 25 mM potassium phosphate (pH 7.2), 5 mM magnesium chloride, 260 µM NADH, 67 µM decylubiquinone (Sigma-Aldrich), 0.1 mM horse cytochrome c (Sigma-Aldrich) and 3,000 U ml⁻¹ superoxide dismutase to ensure that cytochrome c remained oxidized^{25 (link)}. The assay volume was 170 µl. Four different conditions with or without cytochrome c and decylubiquinone were tested (Supplementary Fig. 5). For each condition, measurements of two blanks (0 µg I + III₂ supercomplex) as well as two measurements each of 2 µg and 4 µg I + III₂ supercomplex were performed. Activity measurements were carried out with a plate reader (Multiscan Sky, Thermo Fisher Scientific) at 340 nm (NADH absorbance; extinction coefficient of NADH: 6.22 mM⁻¹ cm⁻¹).

The extracellular proline concentrations after a 24 h incubation of cultured astrocytes with proline was determined by a modification of a colorimetric assay that uses the formation of a specific proline-ninhydrin condensation product under acidic conditions at high temperature [35 (link)]. Briefly, samples of IB that had been collected after the incubation of cells with proline were diluted with IB. 100 µL of the dilution (or of proline standards in IB in concentrations between 0 and 200 µM) were mixed with 200 µL of glacial acetic acid containing 1.25% (w/v) ninhydrin and subsequently incubated at 100 °C for 30 min in a thermoblock (MBT 250, Kleinfeld Labortechnik, Gehrden, Germany). After cooling to room temperature, 200 µL of the reaction mixtures were transferred to wells of a microtiter plate and the absorbance of the generated proline-ninhydrin condensation product was determined at 514 nm in a microtiter plate photometer (Multiscan sky, Thermo Fisher Scientific, Schwerte, Germany). Proline concentrations in samples were calculated by making use of the linear calibration curve generated from the absorbances obtained for the proline standards.

Exosomes were quantified on protein content bases using the Bradford standard protein assay. We diluted 2.0 µL of the samples in 38 µL of deionized distilled water to obtain a final volume of 40 µL in a 96-well plate. Then, 10 µL of the dilute samples were placed in each well of 96-well plates, and 190 mL of 1 × Bradford assay reagent was added (Quickstart, Bradford Protein Assay Kit 1, BioRad, Hercules, CA, USA) and incubated for 5 min. Next, the OD value was recorded using a microplate reader (Multiscan Sky, Thermoscientific) at 595 nm wavelength. Bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) 50 µg/µL was run as the standard control.