In each experiment, each condition was performed in duplicate. Immunohistochemistry and analysis Immunohistochemistry was performed while described previously , about 10 m frozen sections of chilly PBS-perfused mind, using monoclonal antibodies specific for the integrin subunits 1 (clone Ha31/8) and 2 (clone Ha1/29), and the pericyte marker 4-Hydroxytamoxifen NG2. Sigma Chemical Co., St. Louis, MO, USA) and endothelial cell growth product (ECGS) (Upstate Cell Signaling Solutions, Lake Placid, NY, USA), on six-well plates coated with type I collagen (Sigma Chemical Co.). To obtain BECs, puromycin (4 g/ml; Alexis GmbH, Grunberg, Germany) was included in the tradition media on days 1 to 3 to remove contaminating cell types. Endothelial cell purity was >99% as determined by circulation cytometry with CD31. For those experiments, BECs were used only for the 1st passage. Pericytes were acquired using the same approach, except the puromycin step was omitted. The pericyte cultures were cultivated in ECGM, with the medium changed every 3 days. On reaching confluency, cultures were harvested with trypsin and passaged. During the 1st two passages, pericyte cultures were cultivated in ECGM, but on the third passage, they were switched to pericyte medium (PCM; ScienCell Study Laboratories, Carlsbad, CA, USA) comprising 2% FBS. In earlier studies we found that, using this approach, cultures of pericytes become highly purified after the third passage, at which point these cultures are more than 99% pericytes as determined by expression of the pericyte marker NG2 and the PDGF- receptor, and contain less than 1% of contaminating endothelial cells (CD31), astrocytes (glial fibrillary acidic protein; GFAP), or microglia (Mac pc-1), as determined by fluorescent immunocytochemistry . All studies were performed on pericytes at passages 4-Hydroxytamoxifen 4 to 8. Pericytes were expanded in PCM comprising 2% FBS, but all practical assays were performed in serum-free DMEM comprising N1 product, L-glutamine, and penicillin/streptomycin (all from Sigma Chemical Co.). Cytokine treatment and antibodies To investigate the influence of cytokines on pericyte behavior and manifestation of integrin subunits, pericytes were cultured on collagen 4-Hydroxytamoxifen I in the presence of 20 ng/ml fundamental fibroblast growth element (bFGF; Invitrogen Corp., Carlsbad, CA, USA), 20 ng/ml platelet derived growth element (PDGF-B) 2 ng/ml transforming growth element (TGF)-1 10 ng/ml tumor necrosis element (TNF)-, or 10 ng/ml vascular endothelial growth element (VEGF) (all R&D Systems, Minneapolis, MN, USA). These concentrations were selected based on the findings of previous studies [26,27]. The following monoclonal antibodies (BD Pharmingen, La Jolla, CA, USA) were used: monoclonal antibodies reactive for the integrin subunits 1 (clone Ha31/8), 2 (clone Ha1/29), 4 (clone MFR4.B), 5 (clone 5H10-27 (MFR5), 6 (clone GoH3), 1 (clone Ha2/5), and Mac pc-1 (clone M1/170); CD31 (clone MEC13.3); and isotype control antibodies: rat anti-KLH (A110-2) and hamster anti-TNP-KLH (G235-1). Additional antibodies used in this study included Cy3-conjugated anti-GFAP (Sigma Chemical Co.) and rabbit anti-NG2 and anti-PDGF- receptor antibodies (both kindly provided by Dr William Stallcup, Sanford-Burnham Medical Study Institute, La Jolla, CA, USA). Circulation cytometry Integrin manifestation by BECs and pericytes (treated with different cytokines for 2 days) was examined as explained previously . Briefly, cells were removed from the six-well tradition plates, and cell-surface manifestation of the integrin subunits 1, 2, 4, 5, 6, or 1 was analyzed by circulation cytometry using phycoerythrin (PE)-conjugated monoclonal antibodies (all BD Pharmingen). The fluorescent intensity of the labeled cells was analyzed with a circulation cytometer (FACScan; Becton Dickinson, San Diego, CA, USA), with 10,000 events captured for each condition. In each experimental condition, the mean fluorescent intensity was compared with the control (no element) condition, and indicated as the percentage switch relative to control. Each experiment was repeated a minimum of four times. Cell-adhesion assays Adhesion assays were performed as explained previously . Briefly, substrates were prepared by covering the central part of glass coverslips in 24-well plates (Nunc; BD Biosciences, San Jose, CA, USA) with 25 l of ECM remedy (10 g/ml of collagen I, collagen IV, fibronectin, HSPG, or laminin-1; all from Sigma Chemical Co.) for 2 hours at 37C. Substrates were washed twice before addition of cells. Pericytes were prepared as explained above, centrifuged, and re-suspended in N1 serum-free press, then 2,000 cells were applied to the substrates inside a 25 l drop and incubated at 37C Rabbit polyclonal to ABHD4 for 1, 4, or 8 hours. In function-blocking experiments, antibodies were included at 5 g/ml. The assay was halted by adding 1 ml of DMEM and washing off any loosely attached cells. Attached cells were fixed in 4% paraformaldehyde in PBS for 20 moments, and stored in PBS. Adhesion was quantified under phase microscopy by counting all attached cells within five fields.