2.4. Percoll Density Gradient Centrifugation (PDGC)

Singly dissociated ovarian cells were suspended in 200 µL DPBS and loaded onto the top of a discontinuous 7-step Percoll (Sigma-Aldrich, St. Louis, MO, USA) solution consisting of 1 mL each of 20%, 25%, 30%, 35%, 40%, 50%, and 60% in a 15 mL conical tube (Falcon, Durham, NC, USA). Each Percoll solution was prepared by the manufacturer’s instruction. After centrifugation at 800× g for 30 min, the cells from each density fraction were carefully harvested, washed twice with DPBS, and then used for experiments. Cell morphologies were observed under a phase-contrast inverted microscope (TS-100F, Nikon, Tokyo, Japan). Morphology of OGSCs was defined as cells harboring a large nucleus with one or two prominent nucleoli [24,25].