Cell culture and intervention protocols
C2C12 murine myoblasts (American Type Culture Collection, CRL-1772) were
cultured in growth medium consisting of Dulbecco’s Modified Eagle’s
Medium (DMEM; Thermo Fisher Scientific, USA) supplemented with 1%
penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL
streptomycin; Thermo Fisher Scientific, USA) and 10% fetal bovine serum
(FBS; Thermo Fisher Scientific, USA). Cells were cultured in a T-75
culture flask at a density of 5 x 103 viable cells/cm2
and were passaged at 70-80% confluence. Flasks were kept in a
humidified incubator at 37°C with the supplementation of 5%
CO2. To induce differentiation, confluent monolayers of
C2C12 myoblasts were cultured in differentiation medium (DM) consist of
DMEM supplemented with 1% penicillin/streptomycin and 2% horse serum
(Thermo Fisher Scientific, USA) and the DM was changed daily. All
experiments were performed on day 6 when most myoblasts have fused to
form mature myotubes.
The gas phase of cigarette smoke, otherwise known as cigarette smoke
extract (CSE), was prepared by bubbling 1 cigarette (Winfield Red,
Phillip Morris International, Australia) through 25 mL of pre-warmed DM
at a rate of 5 mL·sec-1 to produce 100% CSE stock
solution. The stock solution was sterile filtered and serially diluted
with pre-warmed DM to obtain concentrations required for
experimentation. Hydrogen peroxide
(H2O2; Chem-Supply, Australia) was
prepared in sterile water resulting in a 3,000 µM stock solution. The
stock solution was serially diluted with pre-warmed DM to obtain the
required concentrations for experimentation. Apocynin (Sigma-Aldrich,
USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) to
give a stock solution of 500µM (stock solution). The stock solution was
diluted with pre-warmed DM to give a final administering concentration
of 500nM (68). To ensure bioavailability, apocynin was pre-incubated for
30 min prior to administration of the respective oxidative insults
(H2O2 or CSE), recapitulating that in
our animal model.