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The human bronchial Microbiology inhibitor epithelial cell (BEC) line 16HB14o- [22] was kindly provided by Dr D. Greunert, University of California, San Francisco. Cells were cultured in T-25 flasks precoated with human fibronectin and bovine collagen (BD biosciences). Cells were cultured in minimal essential medium supplemented with 10% fetal calf serum (exosome depleted at 100?000?g overnight), glutamine (200?mM), and penicillin (50?mg/ml) and kept at 37��C with 5% CO2. After reaching confluence, cells were detached using Trypsin�CEDTA solution (R&D Systems, Minneapolis, MN). BALF exosomes corresponding to 1.5?ml of original BALF were added to 50?000 BEC in 250?��l medium in 24-well plates in duplicate (48?h). Supernatants were stored at ?20��C before analyzed for cytokine and LT content as described below. Viability was controlled by estimating confluence, which CAPNS1 always exceeded 90%. IL-6 and IL-8 contents in supernatants were analyzed by ELISA in duplicate according to manufacturer's instructions (Biolegend, San Diego, CA). Levels of LTs were quantified by reverse-phase HPLC (RT-HPLC) as described [23]. Exosomes (25?��g) were incubated in PBS with 20?��M LTA4 for 5?min at 37��C. Reactions were stopped by adding 100% methanol containing 250?pmol PGB2 as internal standard, and chilling on ice. Samples were incubated at ?20��C overnight and centrifuged (4��C, 10?000?g, 10?min). Supernatants were analyzed by RP-HPLC for LTC4 and LTB4 as described [24]. Because of occasional decrease in viability during the culture of frozen cells, the culture protocol was slightly changed for blocking experiments. BEC (25?000/250?��l medium/well) were first cultured alone on fibronectin-/collagen-precoated (BD bioscience) 48-well plates at 37��C for 24?h. New medium and BALF exosomes (1?��g/well) were added in duplicate with or without the CysLT1 receptor antagonist Montelukast (100?nM, Cayman Europe). Supernatants were collected after 24?h and analyzed for IL-8 as described above. Mann�CWhitney Selleckchem Talazoparib U-test was used for comparison between controls and asthmatics, and Wilcoxon matched-pairs signed-ranks test for before and after allergen challenge and Montelukast blocking experiments. P-values were considered significant at P?
