Cells of the lung, adipose tissue and liver were isolated by enzymatic digestion as follows:
Lung: Lungs were perfused with PBS from the heart, isolated and minced first with scissors, followed by the gentleMACS program m_lung_01-02, digested 30 min at 37 °C on an orbital shaker with 0.15 WünschU/mg Liberase (Cat# 5401020001, Roche) and 0.1 mg/mL DNase I, and homogenized by using gentleMACS program m_lung_02_01. Lung cells were enriched for leukocytes by Percoll gradient (40% /70%, 600 g, 20 min, minimal brake). Gating see Additional file1 : Figure S2A.
Adipose tissue: Epididymal adipose tissue was minced and digested with 1.5 mg/mL collagenase IV (Cat# LS004189, Worthington), 10 mM HEPES and 8.25 µg/mL DNase I for 20–25 min on a thermomixer with 400 rpm. Erythrocytes were removed by red cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Adipose tissue macrophages were gated as CD45+F4/80+CD11b+ and subdivided into subpopulations double negative (DN), pro-inflammatory M1a (CD11c+CD206−) and M1b (CD11c+CD206int), and anti-inflammatory M2 (CD11c− to lowCD206+). Gating see Additional file1 : Figure S2B.
Liver macrophages: One liver lobe was minced and digested using 1.5 mg/mL collagenase IV, 10 mM HEPES and 8.25 µg/mL DNase I, on a thermomixer with 400 rpm. After 15 min, the tissue was mechanistically dissected by pipetting up and down with a 1 mL pipette, and digested for another 15 min, followed by filtration and Percoll gradient. Gating see Additional file1 : Figure S2C.
Cell analysis was performed on a FACS LSRII Fortessa (BD Biosciences). Acquired data were analyzed using FlowJo software (Version 9.9 or higher), TreeStar Inc. Ashland, OR, USA).
From Biolegend, we obtained antibodies against CD11b (M1/70), CD11c (N418), MHCII (M5/114.14.2), Ly6C (HK1.4), CD45 (30-F11), F4/80 (BM8), CD103 (2E7), CD24 (M1/69), CD64 (X54-5/7.1), CD3 (145-2C11), CD19 (6D5), NK1.1 (PL136), Ly6G (1A8) and CD206 (C068C2). mAb for CCR2 (475,301) was purchased from R&D. mAb for Siglec F (E50-2440) was obtained from BD. For further details see Additional file1 : Table S3.
Lung: Lungs were perfused with PBS from the heart, isolated and minced first with scissors, followed by the gentleMACS program m_lung_01-02, digested 30 min at 37 °C on an orbital shaker with 0.15 WünschU/mg Liberase (Cat# 5401020001, Roche) and 0.1 mg/mL DNase I, and homogenized by using gentleMACS program m_lung_02_01. Lung cells were enriched for leukocytes by Percoll gradient (40% /70%, 600 g, 20 min, minimal brake). Gating see Additional file
Adipose tissue: Epididymal adipose tissue was minced and digested with 1.5 mg/mL collagenase IV (Cat# LS004189, Worthington), 10 mM HEPES and 8.25 µg/mL DNase I for 20–25 min on a thermomixer with 400 rpm. Erythrocytes were removed by red cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Adipose tissue macrophages were gated as CD45+F4/80+CD11b+ and subdivided into subpopulations double negative (DN), pro-inflammatory M1a (CD11c+CD206−) and M1b (CD11c+CD206int), and anti-inflammatory M2 (CD11c− to lowCD206+). Gating see Additional file
Liver macrophages: One liver lobe was minced and digested using 1.5 mg/mL collagenase IV, 10 mM HEPES and 8.25 µg/mL DNase I, on a thermomixer with 400 rpm. After 15 min, the tissue was mechanistically dissected by pipetting up and down with a 1 mL pipette, and digested for another 15 min, followed by filtration and Percoll gradient. Gating see Additional file
Cell analysis was performed on a FACS LSRII Fortessa (BD Biosciences). Acquired data were analyzed using FlowJo software (Version 9.9 or higher), TreeStar Inc. Ashland, OR, USA).
From Biolegend, we obtained antibodies against CD11b (M1/70), CD11c (N418), MHCII (M5/114.14.2), Ly6C (HK1.4), CD45 (30-F11), F4/80 (BM8), CD103 (2E7), CD24 (M1/69), CD64 (X54-5/7.1), CD3 (145-2C11), CD19 (6D5), NK1.1 (PL136), Ly6G (1A8) and CD206 (C068C2). mAb for CCR2 (475,301) was purchased from R&D. mAb for Siglec F (E50-2440) was obtained from BD. For further details see Additional file
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