Fig. 1.

Schematics of the cellular assays performed to examine the effect of prolactin upon proliferating and differentiating hippocampal progenitor cells (HPCs). Schematics indicate the timing of treatments, and length of proliferation and differentiation phases in days. (A, B) Cells are cultured in media containing 4-OHT and growth factors (EGF and bFGF) for the duration of the proliferation phases (first 72 h). After 24 h cells are treated with either prolactin, estradiol or testosterone. After 24 h or 48 h cells are fixed and processed for ICC and quantification of cellular markers. (C, D) After the initial proliferation phase, cells undergo two full media washes with differentiation media (lacking EGF, bFGF and 4-OHT) and are cultured for the remaining assay tie in differentiation media containing treatment. (E) Three days after the initiation of differentiation, cells are supplemented with an additional hormone treatment to ensure prolactin levels remain at the target concentration throughout the differentiation phase. (E) Representative images of HPCs allowed to differentiate for 7 days, illustrating the ability to produce DCX and MAP2-positive neurons and S100β-positive astrocytes. DAPI nuclear staining shown in blue. Scale bar = 100 μm.