ATM isolation and analysis
Mice were euthanized chemically and epididymal fat was processed for isolation of ATM. 1 gram of adipose fat was rinsed in PBS and minced to small pieces in HEPES-DMEM buffer containing 10 mg/ml BSA. The suspension was centrifuged at 1000 g for 10 min and the resultant supernatant was pipette off to fresh tubes. 1 mg/ml of collagenase type-IV and 50 U/ml DNAse-II were added to this suspension and incubated at 37 °C for 45 mins with moderate shaking, filtered through 250-micron filter and the resultant solution was centrifuged again at 1000 g for 10 mins. Floating cells contained adipocyte and pellet are SVC. RBC lysis buffer was added gently to disrupt the sedimented pellets and centrifuged at 1000 g for 10 min at 4 °C. Fat macrophage cells were isolated by using BD IMag anti-mouse CD11b + magnetic beads through positive selection under the magnetic field. The percentage purity of macrophage isolation was determined by FACSCANTO II flow cytometer using APC tagged CD11b monoclonal antibody. The isolated macrophage was processed for RNA isolation, cDNA synthesis and various M1-M2 markers were evaluated using real-time PCR.