Biology Reference
In-Depth Information
Hydrogen Peroxide treatment
F3 and F3.Akt1 cells were plated 1×10
4
cells per well in 96 well-plates (Falcon,
Becton Dickinson, Franklin lakes, NJ) with DMEM containing 5% FBS and
incubated for overnight. Hydrogen peroxide (Sigma) was added to each well con-
taining F3 and F3.Akt1 cells to give a final H2O2 concentration in the range
from 0 to 0.5 mM. Control wells contained normal medium. Cells were left for 6
hr and 24 hr for viability assay and for 6 hr for western blot analysis.
Cell Viability Assay
Cell viability was determined by the conversion of MTT [3-(4,5-dimethylthi-
axol-2-yl)-2,5 diphenyl tetrazolium bromide] to formazan utilizing NADH and
NADPH pyridine nucleotide cofactors. After stimulation with H2O2 at differ-
ent dose for 6 or 24 hr, MTT [2 mg/ml in phosphate buffered saline (PBS)]
was added to each well and incubated for 2 hr. The media containing MTT was
removed, 100 µL of dimethyl sulfoxide (DMSO, Sigma) added to each well and
absorbance read at 570 nm.
Western Blot Analysis
Western blot analysis was performed with 50 µg total protein extract separated on
10% SDS-PAGE gels that were subsequently transferred to polyvinylidene difluo-
ride (PVDF) membrane (Millipore, Billerica, MA). Blocking of membranes with
5% skim milk in TBST and washed in TBST, membranes were incubated with
anti-phospho-Akt1-Thr 308 (1:500, Upstate), anti-caspase-3 (1:1000, Chemicon)
and anti-beta-actin antibody (1:10,000, Santa Cruz) at 4°C overnight, membrane
washed in Tris-buffered saline containing 0.05% tween 20 for 1 hr at RT, then
incubated in secondary antibody [horseradish peroxidase-conjugated anti-rabbit
or anti-mouse IgG] (Sigma) for 2 hr at RT. Immunoreactive bands were detected
by chemiluminescence using ECL system. Western blots analyses were performed
on samples from twice separate experiments.
Mouse intracerebral Hemorrhage stroke Model
All experimental procedures were approved by the Animal Care Committee of
the University of British Columbia. ICH was induced by stereotaxic, intrastri-
atal administration of bacterial collagenase by previously described methods [12],
[16], [17]. In brief, after an intraperitoneal injection of 1% ketamine (30 mg/
kg) and xylazine hydrochloride (4 mg/kg), the mice were placed in a stereotaxic
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