Osteopontin Regulation of Mouse Brain Endothelial Cells

Mouse brain microvascular endothelial cells (MBMEC) at 3 days post isolation were seeded in 24-well plates at passage 1. These cells were treated with recombinant osteopontin (R&D Systems) with or without anti-osteopontin IgG (R&D systems) at the indicated concentrations at 48 h post plating when the cells reached confluency. The treatment period was for 24 h followed by harvesting the cells to obtain RNA and cDNA exactly as described previously^{12 (link)} using RNeasy micro kit (Qiagen) followed by cDNA synthesis (Revertaid kit, Thermofisher). qRT-PCR was performed using Absolute qPCR SYBR Green Fluorescein Mix in IQ5 instrument (Biorad) using a previously established PCR protocol^{12 (link),32 (link),38 (link)}. Rplp0 was used as a house-keeping gene and qRT-PCR was performed for Spp1, Cd44 and Cdh5 using 2^−ΔCt method. Primers are listed in Supplementary Table S3.