Brain vascular pericytes

Human astrocytoma cell line CCF-STTG1 (cat# 90021502) acquired from European Collection of Authenticated Cell Cultures (ECACC) were expanded at AstraZeneca and used as the parental line to generate an AXL−/− line by CRISPR-Cas9 gene editing and polyclonal AXL-reconstituted CCF-STTG1 lines (described below). Human microglia clone 3 (HMC3, cat# CRL-3304) was acquired from American Type Culture Collection (ATCC). Immortalized LXR double knockout (LXRα−/LXRβ-) and LXR expressing (LXRα+/LXRβ-) mouse embryonic fibroblasts (MEFs) were kindly provided by Dr. Peter Tontonoz (California, USA) [36 (link)]. Primary human astrocytes (cat# 1800) and brain vascular pericytes (cat# 1200) were acquired from ScienCell. Human APOE3 targeted-replacement mice [37 ] were obtained from the Cure Alzheimer Fund and primary murine mixed glia were cultured from postnatal day 0–2 pups as described [38 ]. The LXR agonist T0901317 was acquired from Sigma-Aldrich (cat# 575310). AZ7235 was synthesized as previously described [35 ]. Commercial Axl inhibitor R428 (cat# 21523) and UNC2025 (cat# 16613) were purchased from Cayman Chemical. Axl inhibitor S49076 was purchased from Selleckchem (cat# S8404). Stocks of all compounds were prepared in dimethyl sulfoxide (DMSO).

Human cervical carcinoma cell (HeLa, catalog no. CRM-CCL-2^TM) and mouse mononuclear macrophages (J774A.1, catalog no. TIB-67^TM) were obtained from the American Type Culture Collection (ATCC). The human brain endothelial cell (hCMEC/D3, catalog no. 337728) was purchased from BeNa Culture Collection. The luciferase-transfected glioblastoma cell lines (U251-luc, catalog no. CBP30207L and GL261-luc, catalog no. iCell-0059a) were maintained in our laboratory (Shenzhen Second People’s Hospital, Shenzhen, China). Human astrocytes (catalog no. 1800) and brain vascular pericytes (catalog no. 1200) were purchased from Sciencell. The cells have been confirmed without mycoplasma contamination by mycoplasma detection kit (Beijing Solarbio Science & Technology Co., Ltd., CA1080). All types of cells were cultured in DMEM and supplemented with 10% FBS and 1% (v/v) penicillin-streptomycin. The normoxic cells were incubated in a humidified atmosphere with 5% CO₂ and the hypoxia cells were maintained in an incubator with 1% O₂, 5% CO₂, and 94% N₂. Red blood cells (RBC) were obtained from the peripheral blood of mice.