Mouse cytokine antibody array

Mouse cytokine antibody array (R&D Systems) was used to screen 40
cytokines. Two hundred μg of protein, Biotin-Conjugated Anti-Cytokines
antibodies and HRP-Conjugated Streptavidin were used as recommended by the
vendor. The arrays were scanned, and images acquired using FluorChem M System
(ProteinSimple) and quantified using ImageJ software (NIH, Bethesda, MD).

To prepare supernatant samples for proteome profiler array, EpRAS cells were seeded in a 35-mm cell culture treated dishes at a density of 0.28 × 10⁶ cells/cm² and treated with 0.5% E-Cig. Non-treated condition was included as a control. After 24 h, the supernatant was collected and analyzed with mouse cytokine antibody array (R&D System, Minneapolis, MN) per manufacturer’s instruction. Briefly, the membrane was treated with blocking buffer for 30 min then incubated with 1 mL of EpRAS supernatant overnight at 4 °C. After this incubation period, the membranes were washed five times with a washing buffer and incubated for 1 h at room temperature with biotin-conjugated antibodies against murine cytokines (1:250 dilution). Thereafter, the membranes were washed five times, incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated streptavidin (1:1000 dilution) and washed five times again. Finally, the reaction was developed in a mixture of SuperSignal West Pico luminol/enhancer and stable peroxide solutions (Thermo Scientific, Rockford, IL) and exposed to BioRad imaging system. The pixel density of each spot was subtracted to that of the correlated negative control (neg)/background and then analyzed with ImageJ software (NIH). The experiment was performed in duplicate.

200 mg epididymal adipose tissue explants were cultured in serum-free AIM V media with AlbuMax and BSA (Life Technologies) to create conditioned media (CM). Confluent 3T3-L1 cells were treated with a 1:1 mixture of CM and MDI differentiation media (0.5mM methylisobutylxanthine, 1μM dexamethasone and 10μg/mL human recombinant insulin in DMEM); after 3 days, media was replaced with another 1:1 mixture of CM and insulin medium (10μg insulin in DMEM). For TNFα neutralizing experiments, CM was treated 1h with 100 ng/mL blocking antibody before using the CM in 3T3-L1 cultures at a final concentration of 50ng/mL (D2H4 mAb, CST). For cytokine and chemokine treatments (10 ng/ml, PeproTech) 3T3-L1 cells were treated during both phases of differentiation. Mouse Cytokine Antibody Array (R&D Systems) was used to evaluate adipose tissue explant conditioned media (48 hours, pooled from 3 mice). Quantitation was performed by ImageJ after background subtraction and normalization.