Exoquick

Plasma was prepared by three centrifugation steps at 300 g for 10 min, 2000 g for 10 min, and 10 000 g for 30 min to remove cells and cell debris. Exosomes were then isolated from 143 plasma samples as well as from supernatants of ovarian cancer cell lines OVCAR3 and SKOV3 by ExoQuick (BioCat, Heidelberg, Germany) according to the manufacturer's instructions. Briefly, 500 μL of plasma and 5 mL cell supernatants were incubated with 120 μL and 1 mL ExoQuick exosome precipitation solution, respectively, at 4 °C for 30 min. Then, continued with a centrifugation at 1500 g for 30 min to remove the supernatants and additional 5 min to remove residual fluid. The precipitated exosomes were resuspended in PBS (phosphate‐buffered saline) buffer (Life Technologies, Darmstadt, Germany).

Total exosomes were isolated by ExoQuick (BioCat, Heidelberg, Germany) according to the manufacturer’s instructions. Briefly, 550 μl of plasma, removed from cells and debris by two centrifugation steps at 3000g for 15 min, was incubated with 120 μl ExoQuick exosome precipitation solution at 4 °C, for 30 min. Following centrifugation at 1500g for 30 min, the exosomes were precipitated and then resuspended in 50 μl PBS (phosphate-buffered saline) buffer (Life Technologies, Darmstadt, Germany).