NRVMs were prepared
from six, 1–3 day old pups, as previously described, with minor
modifications.24 (link),25 (link) The University of Colorado Denver
Animal Care and Use Committee’s regulations were fulfilled
for all animal studies. Using scissors, the ventricles and atria were
separated, and the ventricles were then separated, dissociated in
calcium-free and bicarbonate-free Hanks with Hepes (CBFHH) buffer
containing Heparin (Sigma-Aldrich) (10 U·mL–1), and digested in a CBFHH solution containing 1.12 mg·mL–1 of trypsine (Gibco) and 20 μg·mL–1 of DNAse (Sigma-Aldrich). Two sequential pre-plating steps, on 100
mm dishes in Dulbecco’s modified Eagle’s medium (Gibco),
were used to enrich cardiomyocytes (>90% purity) over non-myocytes.
First, 4.5 g augmented with 5% bovine calf serum (Gibco) and then
2 mg mL–1 vitamin B12 (Sigma-Aldrich) and cultured
unattached cells, predominantly myocytes, were harvested and cultivated
in gelatin-coated dishes before being given various treatments and
further examinations. A minimum of three separate cell isolations
were used to test each experimental condition in triplicate.
from six, 1–3 day old pups, as previously described, with minor
modifications.24 (link),25 (link) The University of Colorado Denver
Animal Care and Use Committee’s regulations were fulfilled
for all animal studies. Using scissors, the ventricles and atria were
separated, and the ventricles were then separated, dissociated in
calcium-free and bicarbonate-free Hanks with Hepes (CBFHH) buffer
containing Heparin (Sigma-Aldrich) (10 U·mL–1), and digested in a CBFHH solution containing 1.12 mg·mL–1 of trypsine (Gibco) and 20 μg·mL–1 of DNAse (Sigma-Aldrich). Two sequential pre-plating steps, on 100
mm dishes in Dulbecco’s modified Eagle’s medium (Gibco),
were used to enrich cardiomyocytes (>90% purity) over non-myocytes.
First, 4.5 g augmented with 5% bovine calf serum (Gibco) and then
2 mg mL–1 vitamin B12 (Sigma-Aldrich) and cultured
unattached cells, predominantly myocytes, were harvested and cultivated
in gelatin-coated dishes before being given various treatments and
further examinations. A minimum of three separate cell isolations
were used to test each experimental condition in triplicate.
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