Thank you Khalid!

Yefei Pang, Peter Thomas,General and Comparative Endocrinology

Preparation of ovarian follicle cell and denuded oocyte fractionsIsolated ovarian follicles were washed several times with 5 ml 60% L-15 medium followed by a 30 min treatment at room temperature with 0.1 mg/ml collagenase (Sigma). The ovarian preparation was subsequently forced through a narrow pipette (∼1 mm diameter) 10–20 times to break up the tissue. The resulting mixture of follicle cells and denuded oocytes was transferred to a new tube and allowed to settle for 1 min. The supernatant containing the follicle cells was then transferred to 1.5 ml tubes and washed 2 times with L-15 medium by centrifugation (1000 rpm, 2 min). The effectiveness of the separation procedure was confirmed by examination of the denuded oocytes and follicle cell suspensions under a microscope, followed by DAPI staining of the oocytes to confirm they were completely denuded of follicle cells. The denuded oocyte and follicle cell fractions were either used for extraction of total RNA immediately, or stored at −80 °C for later analysis.