Sucrose Gradient Fractionation of Cell Organelles

Sucrose gradient was performed as described elsewhere [51 (link)]. Briefly, two 10 cm dishes of A673 and TC71 cells were washed twice with cold PBS and resuspended in 500 μl of homogenization buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L sodium chloride, and 5 mmol/L EDTA), supplemented with 10 μg/mL leupeptin and 10 μg/ml aprotinin. Cells were disrupted at 4°C by nitrogen cavitation in a cell disruption bomb (Parr Instrument Company) at 800 psi for 15 minutes and collected dropwise. Afterward, cells were passed back and forth through a 22-gauge needle 25 times at 4°C. Nuclei and unbroken cells were removed by centrifugation at 1,600 g in a MLA-130 rotor (Beckman Coulter) for 5 minutes at 4°C. The resulting supernatant (500 μL) was mixed with an equal volume of 2.5 M sucrose and loaded at the bottom of a discontinuous sucrose gradient formed by layers of 200 μL of 30, 25, 20, 15, 10, and 5% sucrose (wt/vol) freshly prepared in homogenization buffer. Gradients were centrifuged in a TLS-55 swinging-rotor (Beckman Coulter) without brake at 166,000 g for 3 h at 4°C. Five fractions of 280 μl were collected from the top in addition to a final lower fraction of 700 μL by using a CentriTube Slicer (Beckman Coulter). The gradients shown in the figures are representative of at least two independent experiments.