High-resolution respirometry (Oroboros oxygraph respiration assay)

Mitochondrial respiration in permeabilised NSCs and hypotonic shock-treated EVs was analysed by high-resolution respirometry (HRR) (Oroboros Instruments, Innsbruck, Austria). HRR analysis was performed based on previously published methods [29,81]. Briefly, a 2 ml oxygraph (Oxygraph-2k; Oroboros Instruments) chamber was washed with 70% ethanol, rinsed 3 times with distilled water, then filled with the respiration medium (Medium A plus 1 mM adenosine diphosphate (ADP), 2 mM potassium phosphate, and 1 mg/ml fatty acid free BSA) to be used in each of the assays. Moreover, 20 × 10⁶ NSCs were resuspended in 2 ml of Medium A (20 mM HEPES (adjusted to pH 7.1 with NaOH or KOH), 250 mM sucrose, 10 mM MgCl₂). For permeabilisation, the cell suspension was incubated with 5 μl of 1% digitonin solution for 1 minute at RT on a tube oscillator. Digitonin was then diluted with 5 mL of Medium A and removed by centrifugation at 1,000 x g for 3 minutes. Finally, the pellet was resuspended in 2.1 ml of respiration medium and added into the chamber.

EVs from 36 × 10⁶ NSCs were isolated and subjected to hypo-osmotic shock as described above. Upon removal of homogenisation Medium A through centrifugation at 10,000 x g for 10 minutes, hypotonic-shock treated vesicles were equilibrated in MAITE medium (25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 0.05 mM EDTA, 5 mM MgCl₂, 10 mM Tris-HCl, 10 mM phosphate, pH 7.4). The suspension was centrifuged at 10,000 x g for 10 minutes, resuspended in 100 μl of MAITE buffer, then diluted up to 2.1 mL with MAITE buffer plus 1 mg/ml fatty acid free BSA.

Once samples were loaded in the chambers, a polyvinylidene fluoride stopper was inserted to generate a closed system with a final volume of 2 mL. Oxygen concentration was recorded at 0.5 Hz and converted from voltage to oxygen concentration using a 2-point calibration. Respiration rates (O₂ flux) were calculated as the negative time derivative of oxygen concentration (Datlab Version 4.2.1.50, Oroboros Instruments). The O₂ flux values were corrected for the small amount of back diffusion of oxygen from materials within the chamber, any leak of oxygen from outside of the vessel, and oxygen consumed by the polarographic electrode. A protocol involving serial additions of selected mitochondrial complex substrates and inhibitors was performed for a comprehensive assessment of mitochondrial function. The following substrates and inhibitors were progressively injected with a Hamilton syringe at the respective concentrations: 5 mM glutamate and 5 mM malate (NADH-linked substrates); 0.1 μM rotenone (complex I inhibitor); 5 mM glycerol-3-phosphate and 5 mM succinate (FADH2-linked substrate); cytochrome c (Cyt c) to compensate for a possible loss due to outer membrane disruption; 20 nM antimycin A (complex III inhibitor); 1 mM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD, complex IV electron donor) and finally 0.1 mM KCN (complex IV inhibitor). Data are shown as OCR pmolO₂ normalised by the EVs produced by 10⁶ NSCs.