1). Contaminating cells such as pericytes and astrocytes were not observed in the porcine brain endothelial cell monolayers under phase-contrast microscope following the use of puromycin www.selleckchem.com/products/mi-773-sar405838.html to purify the cultures. Confluent monocultures of porcine brain endothelial cells have an elongated cobblestone-shaped morphology, although not generally so clearly spindle-shaped as reported for rat and bovine brain endothelial cell cultures. Cultures of passage 1 (P.1) PBECs formed confluent monolayers of non-overlapping contact-inhibited cells. Immunocytochemical studies revealed clear marginal staining for occludin and claudin-5 (Fig. 2 A and B respectively) consistent with well-organised

tight junctions, characteristic of the BBB. Clear staining selleck chemicals llc for occludin and claudin-5 (Fig. 2, C and D respectively) was also seen in freshly isolated porcine brain microvessels. P.1 PBECs from the ‘60s’ fraction gave higher TEER than the ‘150s’ fraction (Fig. 3) and were used for all experiments described here. P.1 PBECs (60s) cultured on Transwell Clear inserts were found to give a maximum

TEER of ∼1300 Ω cm2 (mean=789±18 Ω cm2, n=91 inserts in 24 independent experiments and a minimum apparent permeability (Papp) to [14C]sucrose of 3.0×10−6 cm/s (mean=6.07±0.32×10−6 cm/s, n=29 inserts in four independent experiments). The quality control (QC) benchmark for permeability was set at a TEER of 500 Ω cm2 and a Papp sucrose of 8×10−6 cm/s. P.1 PBECs always achieved these targets when the strict preparative methodology was followed, including following the QC benchmarks for morphology and confluence level ( Table 1). TaqMan real-time RT-PCR analysis confirmed the expression of occludin and

claudin-5 in P.1 PBECs. When normalised against GAPDH, mRNA expression level was significantly higher for claudin-5 than for occludin (Fig. 4A). P-glycoprotein (P-gp, ABCB1) is an efflux transporter located on the luminal membrane of the endothelial cells of the BBB. Uptake of [3H]colchicine, a P-gp substrate, into confluent P.1 PBECs is shown in Fig. 5. Addition of 50 μM P-gp inhibitor verapamil to the incubation medium caused a significant increase (p<0.05) in colchicine uptake into P.1 tuclazepam PBECs compared to control cells, evidence for presence of functional P-gp. TaqMan real-time RT-PCR assays confirmed the presence of the efflux transporter breast cancer-resistance protein (BCRP) in P.1 PBECs. Normalisation against GAPDH mRNA expression levels in P.1 PBECs showed that BCRP expression is significantly higher than occludin (p<0.0001) and lower than claudin-5 (p<0.0001; Fig. 4A). The mRNA transcript level of BCRP in P.1 PBECs was twice that of GAPDH (p<0.01; Fig. 4B). Alkaline phosphatase (ALP) activity of P.1 PBECs was measured using p-nitrophenyl phosphate (pNPP) as substrate. Significantly higher levels (p<0.0001) of ALP activity were detected in P.