Obesity drives depot-specific vascular remodeling in white adipose tissue
Obesity drives depot-specific vascular remodeling in white adipose tissue
All animal procedures were performed in accordance with institutional and national regulations and approved by local committees for animal experimentation (RP Karlsruhe, DKFZ). Animals were housed under specific pathogen-free barrier conditions at 21 ± 2 °C with 60 % humidity and a 12-hour light /dark rhythm. Wild type C57BL/6J mice were purchased from Janvier Labs. Diet induced obesity: C57BL/6J male mice were put on a high fat diet containing 60% fat, 20% carbohydrate, 20% proteins (D12492i, Research Diets) or a control diet with 10% fat, 70% carbohydrate, 20% proteins (D12450Bi, Research Diets), starting from 6-8 weeks of age for indicated amount of time. Esm1:Cre-ERT2 x Rosa26-mtdtomato-mEGFP (mTmG) reporter mice: Heterozygous Esm1-CreERT2 Cre+ x homozygous Rosa26 mTmG male mice were put on a control or high fat diet starting from 6-8 weeks of age. After 6 weeks of diet, mice were treated with tamoxifen (100mg/kg body weight) in peanut oil by oral gavage for five days consecutively. After 8 weeks of diet in total mice were sacrificed to collect organs.  Inguinal fat pad representing the subcutaneous white adipose tissue (sWAT) and epididymal fat pad representing the visceral WAT (vWAT) were dissected from lean and obese mice. Prior to mincing the tissue, lymph nodes were removed from sWAT to avoid any contamination from lymphoid structures. Minced tissue was digested with Collagenase II (sWAT 2mg/ml, vWAT 1mg/ml; Gibco) and Dispase II (sWAT 2mg/ml, vWAT 1mg/ml; Sigma) in PBS containing 1% fatty acid free bovine serum albumin (FA-BSA) at 37°C for 45 - 60 mins with rigorous shaking every 10 mins. The digestion was quenched with PBS containing 1% BSA and 5mM EDTA (FACS buffer) and the homogenate was filtered through a 100 µm cell strainer and centrifuged at 300 x g for 5 mins. The supernatant was aspirated and pellet was resuspended in RBC lysis buffer for 1 min. Cells were rinsed in FACS buffer and filtered through a 40 µm filter and resuspended in 1ml FACS buffer. Cells were counted to optimize antibody concentration in the next step. Cell Hashing was performed according to manufacturer's recommendation. The following Hashtag antibodies were used. TotalSeq™- A0301 anti-mouse Hashtag 1 Antibody (Biolegend Cat# 155801), TotalSeq™- A0302 anti-mouse Hashtag 2 Antibody (Biolegend Cat# 155803), TotalSeq™- A0303 anti-mouse Hashtag 3 Antibody (Biolegend Cat# 155805) to label cells from each biological replicate (n=3). Cell Hashing antibody staining was performed simultaneously in the same step as the staining for FACS. The following FACS antibodies were used; CD45-FITC (BD Cat# 553079; 1:200), CD31-APC (BD Cat# 561814; 1:100), F4/80-PE (BD Cat# 565410; 1:100). Cells were incubated in the antibody cocktail for 30 mins on ice and incubated for 5 mins with DAPI. Cells were washed 3 times and resuspended in FACS buffer without EDTA. At this stage cells derived from biological replicates were pooled. Cells were sorted with BD FACS Aria cell sorter equipped with a 100 µm nozzle. All compensation was performed using compensation beads for single color staining and cells for negative and DAPI staining. Cells were gated to collect CD31+ CD45- F4/80- population after excluding debris, dead cells and doublets.