Ultra pure lps ek

Immortalized human fetal astrocytes (Applied Biological Materials Inc.) were seeded onto rat tail collagen type I-coated plates (15 μg/ml, Merck Millipore) at 5000 cells/cm² and maintained in Prigrow IV medium (Applied Biological Materials Inc.) supplemented with 10% heat-inactivated FBS (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37 °C in 5% CO₂. The medium was replaced every other day. At 80% confluence, cells were treated, in triplicates, with TNF (Enzo Life Sciences) (100 ng/ml), IL-1β (Enzo Life Sciences) (100 ng/ml), and ultra-pure LPS-EK (InvivoGen) (10 μg/ml) for 24 h or as indicated. Cells were detached using StemPro Accutase (Gibco), washed three times with ice-cold phosphate-buffered saline (PBS, Gibco), and dry-stored at − 80 °C.

Primary human brain microvascular endothelial cells (HBMECs) (ScienCell) were grown in complete endothelial cell growth medium (EGM-2MV) (Lonza) while primary human astrocytes (ScienCell) were maintained in astrocyte medium (ScienCell). The in vitro BBB model was established as described here [27 (link)]. Transwell PET membrane inserts (12 mm diameter, 0.4 μm pore size) (Corning) were coated with rat tail collagen type I (15 μg/ml) (Merck Millipore) and fibronectin (30 μg/ml) (Sigma-Aldrich) on the apical side and with poly-l-lysine (20 μg/ml) (ScienCell) on the basolateral side. Inserts were turned upside down, and primary human astrocytes were seeded at a density of 25,000 cells per insert and let adhere overnight. Subsequently, inserts were reversed and HBMECs were seeded, on the apical side, at a density of 200,000 cells per insert. The culture was maintained at 37 °C in 5% CO₂ for 4 days.
Ultra-pure LPS-EK (100 ng/ml final concentration) (InvivoGen) was added into the apical chamber. Upon 24-h exposure, astrocytes were detached from the basolateral side of the Transwell insert using Accutase (Gibco), washed three times with ice-cold PBS (Gibco), and dry-stored at − 80 °C. All the experiments were performed in triplicates.