Af594 conjugated transferrin

N-methyl-D-Aspartate (NMDA), NMDA receptor inhibitor, MK801 (Tocris), PKC inhibitor, Staurosporine (Selleckchem), CaMKII inhibitor, KN-93 (Merck-Millipore) were purchased from manufacturers. Fluo-4 AM, AF594-conjugated Transferrin (Thermoscientific), FITC-ChromPure Mouse Transferrin (Jackson Immunoresearch), and IRDye 800CW 2-DG Optical Probe (Li-Cor) were purchased from manufacturers. Anti-GFAP (Cat# 12389S), NeuN (Cat# 24307S), Caveolin-1 (Cat# 3238) antibodies, Rab5 (Cat# 3547S) were purchased from Cell Signaling Technology. Anti-CD31 antibody (Cat# 550,274, BD Bioscience), Anti-Glucose transporter 1 (GLUT1, Cat# MA1-37,783, Invitrogen), Clathrin Heavy Chain antibodies (Cat# MA1-065, Invitrogen), Anti CD13 (Cat# ab7417, Abcam), and LAMP1 (Cat# sc20011, Santa Cruz Biotechnology) were purchased from respective manufaturer. Dilutions for all antibodies were 1:500. Human primary brain endothelial cells were purchased from Cell Systems (Cat # ACBRI 376). Cells were isolated from microvessels from brains of normal, healthy donor and are positive for CD31, vWF. Experiments were done cells with passage number less than 8 for the maintenance of their properties.

To test the effect of the NMDA receptor signaling on the permeability of the brain endothelial cells, we have cultured primary brain endothelial cells on the transwell insert with porous membranes (0.4 μm, Corning, Cat # 353,493) pre-coated with attachment factors (4Z0-201, Cell Systems). Cells were treated with the NMDA (25 μM) or NMDA and MK801 (25 μM and 1 μM, respectively) along with 10 μg/ml of AF594 conjugated transferrin (Cat # T13343, Thermofisher) for upto 15 min. At different time points, media at the bottom chamber was collected. Fluorescence intensity of the AF594 conjugated transferrin that crossed the membrane were measured with excitation at 590 nm and emission at 617 nm with a Flex-Station 3 (Molecular Devices, CA, USA).