Primary HBMECs were obtained from Cell Systems (ACBRI 376). Cells were thawed and plated into T75 flasks for expansion in Lonza EGM2-MV medium (CC-3202) to reach a P4 culture. Cells were subcultured until confluent, passaged at a 1:4 ratio into T75 flasks for P5 cultures, and allowed to expand until confluent prior to freezing (EGM2-MV plus 10% DMSO). Fresh vials were thawed to obtain P6 cultures, which were used for all further experiments.

Primary human astrocytes were obtained from ScienCell Research Laboratories (ScienCell, 1800). Cells were thawed and plated into a T75 flask for expansion in astrocyte medium (AM) (ScienCell, 1801). Cells were subcultured until near confluence (80%–90%), passaged at a 1:4 ratio into T75 flasks, and allowed to expand until near confluence prior to freezing (AM plus 10% DMSO). Fresh vials were thawed and used for all further experiments.

Primary mouse astrocyte cultures were prepared as described previously (20) and purified using negative selection by magnetic CD11b beads (Miltenyi Biotec, 130-049-601). Primary HBMECs were cultured on 100 μg/mL fibronectin and Col IV–coated 24-well or 48-well plates. Cells were seeded at a density of 2.5 × 105 cells/cm2. Confluent cells were treated with IL-1β (10 ng/mL, R&D Systems, 201-LB-005), C3a (500 nM, R&D Systems, 3677-C3-025), C5a (250 nM, R&D Systems, 2037-C5-025), ionomycin (10 μM, Cayman Chemical, 10004974), or in combination with one of the inhibitors SB290157 (5 μM, Calbiochem, 559410), W7 (50 μM, Tocris, 0369) or BAPTA-AM (1 μM, Tocris, 2787). Cells were analyzed after 2 hours or 24 hours of treatment.

TEER analysis was performed using combinations of primary HBMECs and primary human or mouse astrocytes in a coculture. Briefly, semipermeable transwell inserts (Corning, 3470) were coated with 100 μg/mL fibronectin and Col IV on the luminal surface for 2 hours at 37°C in PBS. The remaining coating solution was aspirated, and the transwells were flipped over and placed into 12-well culture plates. The abluminal surface was coated with poly-d-lysine (PDL) at 37°C for 2 hours, and the remaining solution was aspirated. While inverted, primary astrocytes were seeded to the abluminal surface at a density of 1.5 × 105 cells/cm2 and allowed to attach for 4–6 hours at 37°C in 100 μL AM (ScienCell). The membranes were then reverted to normal position in their original culture plate, and the astrocytes were cultured in AM placed into the tissue culture plate (abluminal). The cells were cultured for 48 hours at 37°C and endothelial cells were seeded in the luminal compartment at a density of 1.5 × 105 cells/cm2 in EGM2-MV. All TEER readings were measured using STX2 chopstick electrodes with an EVOM2 volt/ohm meter (World Precision Instruments). Cultures matured over 3–4 days, and when TEER stabilized (~160–180 Ω), treatments were added and TEER was monitored over 24 hours. All TEER readings were normalized to the average reading from 2 cell-free inserts for each time-point recording prior to normalization to the control samples.

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