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The reaction was allowed to proceed for 30 s and was terminated by adding 80 L of a methanolic quenching solution, which consisted of 375 L of 100 mM Tris buffer (pH 9.5) with 0.2 mM EDTA and 625 L methanol

The isolated cumulus cells were then resuspended in Histopaque 1077 (Sigma-Aldrich, Waltham, MA, USA) and enriched with a culture medium containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 5 mg/L insulin, 5 mg/L transferrin, 5 g/L sodium selenite (ITS, Sigma), and 1.25 M androstenedione (4-androstene-3, 17-dione, Sigma)