Wst 1 proliferation assay kit

Inguinal sWAT was dissected from Tc1^−/− and wild type mice, minced and digested using 0.5% collagenase (Sigma Aldrich, St. Louis, MO) at 37 °C for 1 h. Cell suspensions were filtered through a cell strainer with 100 μm nylon mesh (BD Biosciences, San Jose, CA), washed twice with phosphate-buffered saline (PBS) by centrifugation for 3 min at 1,500 rpm at 4 °C. Cell pellets were suspended in red blood cell lysis buffer (Sigma Aldrich) and incubated for 5 min. Cells were washed and centrifuged twice in PBS, and incubated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA) with 10% fetal bovine serum (FBS, Gibco), and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA) at 37 °C in 5% CO₂. The media were replaced after 24 h, and every 3 days thereafter. Cultures were incubated for 4~7 days until 70~80% confluence was attained. ADSCs were passaged and replated at initial concentration of 5 × 10⁵ cells per 100 mm dish. All experiments performed using passage 2 ADSCs. Cell proliferations were analyzed using WST-1 proliferation assay kit according to the manufacturer’s instruction (Roche, Mannheim, Germany). In each experiment, 4 × 10³ cells/well were plated in 96-well plates, and the proliferation was measured in quadruplicate at 0, 24, 48, 72, and 96 h.

The following reagents were used: Dulbecco’s Modified Eagle Medium (DMEM) (GE Healthcare Life Sciences, Logan, UT); Fetal Bovine Serum (FBS) (Atlanta Biologicals, Lawrenceville, GA); penicillin-streptomycin (Invitrogen, Carlsbad, CA); WST-1 proliferation assay kit (Roche, Indianapolis, IN); High-Capacity RNA-to-cDNA™ Kit and SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA); Diff-Quick cell staining kit (Dade Behring, Inc., Newark, DE); anti-human IL-8 ELISA Kit (R&D Systems Inc., Minneapolis, MN); Gemcitabine (Sigma-Aldrich, St. Louis MO). Antibodies used were: anti-CD45, -CD68, -F4/80, -TGF-β1, -IL-8 (Abcam, Cambridge, MA), anti-arginase-1, anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-β-actin (Sigma-Aldrich). Western blotting SuperSignal West Femto Maximum sensitivity substrate kit was purchased from Thermo Scientific (Logan, UT). Immunohistochemical analysis reagent EZ-Dewax (Biogenex, Fremont, CA); background sniper, polymer, and probe were purchased from Biocare Medical (Concord, CA).