Re prepared by resuspending the cell suspension in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, 11995065) supplemented with 10 heat-inactivated fetal bovine serum (FBS, Invitrogen, 16000-044), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 plated at a density of 20 ?106 cells/flask in 75 cm2 cell culture flasks. Cell medium was replaced every 5 days, and after the first medium change, macrophage colony stimulating factor (Invitrogen, PHC9504) at a concentration of 0.25 ng/ml was added to the flasks to promote microglial proliferation. When confluent (7 to 10 days), mixed glial cultures were subjected to shaking at 37 at 220 rpm for 2 h, to promote microglia detachment from the flasks. The cell medium, containing the released microglia cells, was collected from each flask and centrifuged at 1000 g for 5 min to collect the cells which were then plated on cell culture plates for all subsequent experiments.Liao et al. Journal of Neuroinflammation (2016) 13:Page 3 ofMouse microglia isolationAnimals were divided into two groups (n = 6/group): (1) saline or (2) cocaine injections. Cocaine was injected at a dose of 20 mg/kg intraperitoneally for 7 days. On the seventh day, 1 h after the last cocaine injection, microglia were isolated from whole brain homogenates by Percoll gradient centrifugation according to previously published reports [19] with slight modifications. Briefly, the brains were homogenized in phosphate-buffered saline (PBS) (pH 7.4) by passing through a 70-m nylon cell strainer. The homogenates were then centrifuged at 600 g for 6 min. Supernatants were removed, and cell pellets were resuspended in 70 isotonic Percoll (GE Healthcare, Uppsala, Sweden) at room temperature. A discontinuous Percoll density gradient was layered as follows: 70, 50, 35, and 0 isotonic Percoll. The gradient was centrifuged for 20 min at 2000g, and microglia were collected from the interphase between the 70 and 50 Percoll A-836339MedChemExpress A-836339 layers. Cells were washed and then resuspended in sterile PBS followed by flow cytometry analysis by gating the myeloid cells for the CD11b + /CD45dim population.BV-2 cell culturefluorescence was measured using a spectrofluorometer set at 485-nm excitation and 530-nm emission.ImmunoprecipitationImmunoprecipitation was performed as described previously [21, 22]. BV2 cells were treated with cocaine (10 M) for 1 h and then lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich). For each sample, 600 g of protein was used for immunoprecipitation. Cell lysates were incubated with TLR2 antibody overnight at 4 followed by incubation with 30 l of protein A/G beads (Santa Cruz, 2003) for 1.5 h at 4 . The mixture was then centrifuged at 12,000 rpm for 1 min, and the cell pellets were rinsed twice with the lysis buffer (1.0 NP-40, 0.5 sodium deoxycholate, 0.1 SDS, 150 mM NaCl, 9.1 mM Na2HPO4, 1.7 mM NaH2PO4) containing proteinase and phosphatase inhibitors. Finally, 30 l of 2 ?western blot loading buffer was added and boiled for 5 min. Then, the protein complexes were detected using 4G10 antibody (Millipore, Cat# ab5320). Input protein (without antibody addition) served as a control to demonstrate that equal amount of total protein was used.Western blottingThe BV-2 immortalized cell line was obtained from Dr. Sanjay Maggirwar (University of Rochester Medical Center, Rochester, NY, USA) and was grown and routinely maintained in DMEM with 10 FBS at 37 and 5 CO2 and used up to passage 20.siRNA transfec.