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.