Tnf clone mp6 xt22

eWAT and liver was isolated from obese WT and IFNAR^−/− mice. eWAT-isolated SVF cells and liver immune cells were stimulated for 4 h with PMA (50 ng/ml; Sigma-Aldrich) and Ionomycin (1 μg/ml; Calbiochem). Briefly, cells were stained with Live/dead stain (Zombie UV Dye; 1:250; Biolegend), B220 (clone RA3-6B2; 1:100; Biolegend), CD45 (clone 104; 1:500), CD11b (clone M1/70; 1:100), F4/80 (clone BM8; 1:100), Gr1 (clone RB6-8C5; 1:100), CD4 (clone GK1.5; 1:50), CD8 (clone 53-6.7; 1:100), IFNγ (clone XMG1.2; 1:100) TNF (clone MP6-XT22; 1:100), and IL-6 clone (MP5-20F3; 1:100) (all ebioscience). Data were collected using a LSR Fortessa flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Cells were isolated as described above from inguinal, abdominal and thoracic mammary glands. Following isolation, cells were resuspended in full medium (RPMI (Seraglob), 10% FBS (Gibco), 0.01 M HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 2 mM Glutamax (Gibco), 1% Penicillin–Streptomycin (Gibco), 1% nonessential amino acids (Gibco), 57.2 μM β-mercaptoethanol (AppliChem)) with GolgiPlug and GolgiStop 1:1,000 (BD) and exposed to Zymosan 50 µg ml^–1 (InvivoGen) or LPS 300 ng ml^–1 (Sigma) or left unstimulated (negative control). The cells were incubated for 6 h at 37°C, washed and stained for surface markers as described above, then fixed with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C, washed with Perm buffer (2% BSA, 0.5% Saponin, 0.0002% sodium azide in PBS), stained with intracellular antibody mix in Perm buffer for 25 min (TNF (clone MP6-XT22 1:400) and proIL-1β (clone NJTEN3 1:200), purchased either from eBioscience or Biolegend), washed with Perm buffer, resuspended in PBS and acquired.