P-GSE42795-2 - P-GSE42795-2
Alginate-encapsulated follicles were placed in individual wells of a 96-well plate containing 100 ul of growth media (alpha minimum essential medium (alphaMEM) supplemented with 10 mIU/ml recombinant FSH (Organon, Roseland, NJ), 3 mg/ml bovine serum albumin (MP Biomedicals, Irvine, CA), 1 mg/ml bovine fetuin (Sigma, St. Louis, MO), 5 ug/ml insulin, 5 ug/ml transferrin and 5 ng/ml selenium) and cultured for 2, 4, 5, 6 or 8 days in a 5% CO2:21% O2 atmosphere. Half of the culture media (50 ul) was exchanged every 2 days and conditioned media stored at -80ºC. To collect follicles at each time point, alginate-encapsulated follicles were removed from growth media, pooled (20-40 follicles per group, n = 3 independent experiments) and transferred into 1 ml of Liebovitz L-15 media containing 10 U/ml alginate lyase (Sigma) for 20 min at 37ºC. Follicles were aspirated, then transferred into microcentrifuge tubes, flash frozen in liquid nitrogen and stored at -80ºC until subsequent RNA isolation was performed.