Methods and compositions for controlling cardiac fibrosis and remodeling
A heart, cardiology technology, applied in the field of control of myocardial fibrosis and remodeling and composition
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[0137] Materials and methods
[0138] Primary cell culture and transfection
[0139] CM and fibroblast isolation from neonatal mice: Neonatal C57B6 mice were sacrificed within 24 h after birth, and beating hearts were harvested. Carefully excise the atria and great vessels and place them on ice in ADS buffer (H 2 O, NaCl 116mM, HEPES 20mM, NaH 2 PO 4 1mM, KCl 5.4mM, MgSO 4 0.8mM, glucose 5.5mM). Mince the heart using a sterile sharp blade and place the heart in 1.5 ml of PIB digestion buffer (ADS buffer; 0.05 mg / ml collagenase type II (Worthington); 1 mg / ml trypsin; Sigma). ml tubes (5-6 hearts per tube). The tissue was incubated with shaking at 37°C for 15 minutes. Then the supernatant was collected in a medium containing complete medium (DMEM 75%, M199 25% ml, penicillin / streptavidin 1x, L-glutamine 1x, horse serum 10%, fetal bovine serum 5%; Gibco ) in the tube. PIB buffer was added to undigested tissue fragments and digestion was repeated twice. Cells were harv...
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