Research Design and Methods:
Drugs and Chemicals: A pharmacological low dose of naltrexone hydrochloride (LDN) obtained from MP Biomedicals (151725) was used.
Cell lines and culture treatment: Murine macrophage (Raw264.7) cell line was cultured in RPMI media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin
Cell viability: Cell viability assay was carried out in Raw264.7 cells using MTT dye (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) as reported earlier32. Cells were seeded in a 96-well plate and allowed to grow overnight. Further cells were treated with varying doses (0, 2, 4, 6, 8, 10, 20, 40 µM) of the LDN for 24 hrs. Post-treatment, 10µl of MTT (5mg/ml stock in PBS) was added to all the wells. The formazan crystals thus formed were solubilized in 200µl DMSO and the absorbance was recorded using (Infinite M200 Pro TECAN).
RNA isolation and gene expression profile: Raw264.7 cultured cells were treated with 5μM of LDN in the presence and absence of LPS. RNA was isolated from the cells using RNA-Xpress reagent (HIMEDIA-MB601) and 1µg of RNA was reverse-transcribed for quantitative RT-PCR analysis. The results were normalized using housekeeping genes 18S (rRNA) and expression was calculated as fold change by normalizing the control values. Primers are provided in supplemental experimental procedures (table S2).
Western Blot Analysis: Raw264.7 cultured cells were incubated with LDN (0 and 5µM) in the presence or absence of LPS. After treatment, cells were lysed in RIPA buffer containing 1% protease- phosphatase inhibitors. Protein concentration was determined by BCA assay reagent as described in the manufacturer’s (Thermo Scientific-23227) manual. Protein was loaded on SDS-PAGE and electro-blotted on to PVDF membranes. The membrane was incubated in 5% milk blocking solution for 2 hrs at room temperature (RT) and probed against primary antibody (1:2000 diluted in TBST; After washing with TBST, the membrane was incubated with HRP conjugated IgG secondary antibody for 2 hrs and visualized by chemiluminescence. A list of antibodies is provided in supplemental experimental procedures (table S2).
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 1,000g for 10 min and the resultant supernatant was pipette off to fresh tubes. 1mg/ml of collagenase type- IV and 50U/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 1,000g 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 1,000 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.