Mta00b

Cytokines (TNF-α, IL1-β, IL-6 and IFN-γ) were measured in the same lung homogenates as TRP metabolites by ELISA kits (MTA00B, SMLB00C, M6000B, DY485 respectively, R & D systems, Minneapolis, USA) according to the manufacturer's instructions.

Lipopolysaccharide (LPS) and SP600125 were purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). The cell culture Medium (DMEM, MEM, and RPMI 1640), fetal bovine serum (FBS), penicillin–streptomycin (10,000 U/ml) solution, and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.).
Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α (MTA00B), IL-6 (M6000B), and IL-1β (MLB00C) were obtained from R&D Systems (Minneapolis, MN, U.S.A.). The primary antibodies for Ufm1 (ab109305), LZAP (CDK5RAP3) (ab157203), NF-κB p65 (ab16502), p-NF-κB p65 (ab86299), ATF2 (ab47476), p-ATF2 (ab32019), c-Jun (ab32137), p-c-Jun (ab32385), anti-GFP antibody (ab290), and β-actin (ab8226) were from Abcam (Cambridge, U.K.). Anti-Flag antibody (F7425) was form Sigma. Anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (ab97040) was also from Abcam.

The levels of several factors such as the tumor necrosis factor-α (TNF-α, MTA00B, R&D Systems Inc., Minneapolis, MN, USA), IL-1β (MLB00C, R&D Systems), interleukin (IL)-6 (M6000B, R&D Systems), and DA (Shanghai Yueyan Biological Technology, Shanghai, China) in the BALF and hippocampal tissue homogenate were detected using the commercially available ELISA kits [24 (link)].

Supernatant was collected from cultured BECs treated with vehicle (DMSO) or l7β-Oestradiol to measure the concentration of TNFα (R&D Systems, MTA00B) and IL-6 (R&D Systems, M6000B) released using ELISA according to manufacturer's protocol. Specifically, assay diluent was added to standards or samples and incubated for 2 hours at RT. Following washing of all samples, respective protein conjugate was added for 2 hours, following further washing and incubation with substrate solution for 30 mins. OD was measured at 540nm and 450nm (subtracted for wavelength correction), and Protein concentrations were determined by a standard curve.

RAW 264.7 cells were seeded in 12-well plates in complete medium and transfected with a miR-451a mimic, miR-451a inhibitor, and respective negative controls (NCs) (350 nM; Sigma-Aldrich, St. Louis, MO, USA) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After six hours, cells were washed with PBS and stimulated with 500 ng/mL LPS (TLR4 ligand) for 48 h. EV/BiNP studies were performed as described above, with the exception of washing cells with PBS and adding EVs/BiNPs (10⁹/mL) three hours after transfection. Cell culture medium was collected, centrifuged (206× g at 4 °C for 10 min), aliquoted, and immediately stored at −80 °C. The medium was thawed on ice and IL-10 (#M1000B, R&D Systems, Minneapolis, MN, USA) and TNFα (#MTA00B, R&D Systems, Minneapolis, MN, USA) ELISAs were performed according to the manufacturer’s instructions. Absorbance at 450 nm was read with a microplate reader (SpectraMax M5, Molecular Devices, San Jose, CA, USA).