Victor Munive, Andrea Santi & Ignacio Torres-Aleman. A Concerted Action Of Estradiol And Insulin Like Growth Factor I Underlies Sex Differences In Mood Regulation By Exercise. Scientific Reports 6, Article number: 25969 (2016) doi:10.1038/srep25969
...Cell cultures and in vitro assays
Endothelial and choroid plexus cell cultures were performed as described60, with minor modifications. Cells were washed with PBS and serum-free DMEM/F12 without red phenol was added. Three hours later, 1 nM 17β-estradiol (Sigma, USA) was added (or equivalent dose of DMSO in control wells) and 2 hours later biotinylated IGF-I (bIGF-I, IbmH, Germany, 0.2 μg/ml). One hour later cells were lysed and processed for WB. For analysis of surface IGF-I receptors, endothelial cells were plated one day prior to staining in order to achieve 60-80% confluency. Cells were washed with PBS and fasted for three hours with serum-free DMEM/F12 without phenol red. Subsequently, 1 nM 17β-estradiol or equivalent dose of DMSO was added. Two hours later, cells were blocked with a 0.1% BSA /PBS at 4 °C for 5 minutes and washed with PBS 2 times (5 min/Wash). Cells were incubated with IGF-IRα conjugated antibody (Neuromics) at 1:20 dilution in 0.1% BSA /PBS in a humidified chamber for 30 min at 4 °C. Then, cells were washed 3 times in 0.1% BSA/PBS (5 min/wash) protected from light at 4 °C. Finally, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, counterstained with Hoetchst at 1:500 in 0.1% BSA/PBS and washed with PBS....