Anti pecam 1 antibody

Mouse brain microvascular endothelial cells (mBMEC) were prepared using a modified protocol already described (Stamatovic et al., 2006 (link); Stamatovic et al., 2003 (link)). Briefly, brains were collected from four to six-week-old C57BL/6 mice, minced and homogenized gently in a Dounce type homogenizer in Hank’s balance solution (HBSS, Thermo Fisher Scientific). Myelin was removed by resuspending homogenates in 18% Dextran suspension (Dextran 60–90,000; USB,) and centrifuging. Red blood cells were removed by centrifuging isolated microvessels in a Percoll gradient (Pharmacia) at 2700 rpm for 11 min. The isolated microvessels were digested by collagenase/dispase (1μg/ml, Roche), and precipitated with CD31 coated magnet beads (Dynabeads, Thermo Fisher Scientific). These vessels were further cultured in growth media. This protocol typically produces primary endothelial cell cultures that are approximately 99% pure (as determined by immunocytochemistry with an anti-PECAM-1 antibody; BD Bioscience).

Mouse brain microvascular endothelial cells (mBMECs) were prepared from 6 to 8 weeks old CD1 mice (Charles River, Portage, MI) using a modified protocol already described (Dimitrijevic et al., 2006 (link); Stamatovic et al., 2009 (link); Stamatovic et al., 2012 (link)). Briefly, microvessels isolated from cerebral cortex were digested in HBSS solution containing 1 μg/ml collagenase/dispase (Roche, Indianapolis, IN), 10 U/ml DNase I (Sigma-Aldrich, St Louis, MO) and 1 μg/ml Na-p-tosyl-l-lysine chloromethyl ketone (TLCK) for 20 min at 37 °C and purified/precipitated with CD31 coated magnetic beads (Dynabeads, Life Technologies, Grand Island, NY). These vessels were cultured in Dulbecco’s Modified Eagles medium (DMEM) supplemented with 10% inactivated fetal calf serum, 2.5 μg/ml heparin (Sigma-Aldrich), 20 mM HEPES, 2 mM glutamine, 1× antibiotic/antimycotic (all from Life Technologies), and endothelial cell growth supplement (BD Bioscience, San Jose, CA) and grown in 6 well plates coated with collagen type IV (BD Bioscience). This protocol typically produces primary endothelial cell cultures that are approximately 99% pure (as determined by immunocytochemistry with anti-PECAM-1 antibody; BD Bioscience). mBMEC in the first passage was used in all experiments.

After isolation of glomeruli, endothelial cells were isolated as described previously (55 ). In brief, isolated glomeruli were initially digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. Mouse kidney endothelial cells (MKECs) were purified from cell suspension using positive selection with anti-PECAM-1 antibody (BD Pharmingen) conjugated to DynabeadsSheep anti-Rat IgG (Invitrogen) using a Magnetic Particle Concentrator (MPC). Purified cells were cultured on gelatin-coated tissue culture dishes until they became confluent. Next, endothelial cells were further purified using Dynabeads coupled to anti- ICAM-2 antibody and cultured on collagen I-coated dishes. Isolated endothelial cells were characterized by immunostaining and real-time PCR analysis for endothelial markers was performed.