Investigating IGF2-Mediated Cell Proliferation

OVCAR5 were seeded in 12-well plates at 5300 cells/cm², allowed to grow for 2 days, and then serum-starved for 24 h (resulting in a final density of 126,000 cells/well). IGF2 was then either spiked directly into conditioned media (which contains cell-secreted IGFBPs, [27 (link)]) or serum-free media was aspirated, cells were rinsed once with PBS, and the IGF2 treatment was added with fresh serum-free media. For experiments with IGFBP3, 15 nM of recombinant human IGFBP3 (Peprotech) was added 30 min prior to treatment with IGF2. All experiments were done with 1 mL of media per well. Cell proliferation was quantified after 24 h of IGF2 treatment using the Click-iT® EdU Alexa Fluor® 488 flow cytometry assay (Life Technologies) according to manufacturer’s instructions. Cells were incubated with EdU for 6 h prior to sample collection and analyzed on an Accuri C6 flow cytometer (BD, Franklin Lakes, NJ). To investigate the effects of IGF2R knockdown and overexpression on IGF2-induced proliferation, transfected cells were plated as described and treated with either vehicle or 1 nM IGF2.