Tissue immune cell isolation

To isolate adipose macrophages, the adipose stromal vascular fraction was collected as previously described (32). Briefly, mice were euthanized by isoflurane overdose and cervical dislocation and perfused with 20 ml PBS through the left ventricle. Epididymal (unless otherwise stated) or subcutaneous adipose depots were collected, minced, and digested in 6 ml of 2-mg/mL type II collagenase (Sigma # C6885 or Worthington #{"type":"entrez-nucleotide","attrs":{"text":"LS004177","term_id":"1321650547","term_text":"LS004177"}}LS004177) for 30 min at 37°C. Digested tissue was then vortexed, filtered through 100 μm filters with cold PBS, lysed with ACK buffer, and filtered through a 35 μm filter. The stromal vascular fraction was plated at 200,000 cells/well for 2-4 hours to allow for adherence for Seahorse and ELISA experiments or stained for flow cytometry.

To isolate blood monocytes, whole blood was lysed in water and 500,000 cells/well were plated for 2 hours for adherence and used for cytokine production.

To isolate peritoneal macrophages, peritoneal lavage was performed. Cold PBS (5 mL) with 5mM EDTA was injected into the abdominal cavity, the abdomen was massaged for 2 min, and lavage fluid was collected from a small incision by transfer pipette. 200,000 cells/well were plated for adherence for 2 hours for Seahorse metabolic analysis and cytokine production.

To isolate liver macrophages, livers were extracted, minced, and digested as above. The liver suspensions were then plunged through 100 μm filters with cold PBS and lysed with ACK buffer. The cell pellet was resuspended in 33% Percoll and overlayed on top of 66% Percoll. The Percoll gradient was centrifuged at 600 x g for 15 min with the break set to zero. The two middle layers of the Percoll gradient were collected in HBSS and centrifuged at 500 x g for 5 min. 200,000 cells/well were plated for 2 hours for adherence and used for Seahorse metabolic analysis and cytokine production.