2.5. Purification and isolation of mitochondria, MAMs and ER from SH-SY5Y cells

The following protocol is based in the one described in Ref. [36] with some modifications. Cell cultures at 80–90% confluence were harvested with trypsin and the pellet washed with 1 ml 1x PBS and centrifuged at 700 g for 3 min. The supernatant was removed, and the pellet resuspended in 500 μl of hypotonic buffer (250 mM Sucrose, 20 mM HEPES pH 7.45, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, Protease inhibitors) and kept on ice for 30 min. Cells were then disrupted by passing them through a 30G needle for 20 times. The solution was centrifuged at 700 g for 10 min at 4 °C and the supernatant transferred to a 2 ml clean tube. The pellet containing nuclei and intact cells was discarded. The collected solution was centrifuged at 10,000 g for 20 min at 4 °C. The supernatant containing the ER was transferred to a clean 2 ml tube and the pellet containing the crude mitochondria was resuspended in 2 ml isolation medium (250 mM mannitol, 5 mM HEPES pH 7.45, 0.5 mM EGTA, 0.1% BSA). 150 μl of the last suspension was kept as the crude mitochondria (c-mito) fraction. The tube containing the ER was filled with hypotonic buffer, centrifuged at 17,000 g for 35 min at 4 °C, and the new supernatant transferred to an ultracentrifuge tube. The tube was filled with hypotonic buffer and centrifuged at 100,000 g for 1 h at 4 °C. The supernatant was discarded, and the pellet resuspended in 150 μl of hypotonic buffer to obtain the ER fraction. The rest of the solution of isolation medium containing crude mitochondria was transferred to an ultracentrifuge tube filled with 30% Percoll with gradient buffer (30% Percoll, 225 mM mannitol, 25 mM HEPES pH 7.45, 1 mM EGTA, 0.1% BSA) and centrifuged at 95,000 g for 30 min at 4 °C. Two bands or fractions are observed at this point, an upper band or light fraction containing the MAMs and a lower band or heavy fraction containing the pure mitochondria. The lower band was collected with a glass Pasteur pipette, diluted in a clean tube with isolation medium and centrifuged at 6300 g for 10 min at 4 °C. The supernatant was discarded, and the pellet resuspended in 150 μl of isolation medium to obtain the pure mitochondria (p-mito) fraction. The previous upper band containing the MAMs was collected with a glass Pasteur pipette, diluted in isolation medium and centrifuged at 6300 g for 10 min at 4 °C. The supernatant was transferred to an ultracentrifuge tube filled with isolation medium and centrifuged at 100,000 g for 1 h at 4 °C. The supernatant was discarded, and the pellet was resuspended in 150 μl of isolation medium to obtain the MAM fraction.