Interscapular BAT was dissected from the mice gavaged with Bacteroides or vehicle for 12 weeks and processed for cell isolation as described previously (Hagberg et al., 2018 (link)). Briefly, 100 mg of BAT was minced carefully using scissors for 3 min. The minced tissue was digested in a 37°C thermal shaker (#0003637; TAITEC, Saitama, Japan) for 30 min with Collagenase Type 1 (#LS004196; Worthington Biochemical Corp., Lakewood, NJ) prepared in 10 mL PBS. The samples were then centrifuged for 2 min at 20 × g, and the pellets (stromal vascular fraction) were washed 2 times with 10 mL PBS containing 2% bovine serum albumin and incubated with an anti-CD16/CD32 antibody (#553142; BD Biosciences) to block Fc receptors. This was followed by staining with the following antibodies: anti-CD45 (#557659; BD Biosciences), anti-F4/80 (#565411; BD Biosciences), anti-CD11b (#563402; BD Biosciences), and anti-Ly6G (#560602; BD Biosciences). Samples incubated with the isotype-matched antibodies were used as controls. Flow cytometric analysis was performed on an Attune acoustic focusing cytometer (Life Technologies, Grand Island, NY) and by using FlowJo software (Tree Star, Inc., Ashland, OR).
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