Isolation and Culture of Adult Mouse Cardiomyocytes

Mouse ventricular myocytes were isolated from male adult C57/Black mouse (8-12 weeks; Charles River) after dissociation with collagenase II (Worthington, Lakewood, NJ) with previously described method (O'Connell et al., 2007). For surface biotinylation experiments, cardiomyocytes were attached to laminin-precoated culture dishes and cultured in primary cardiomyocyte medium (ScienCell) in 37°C and 5% CO2 incubator overnight in the presence of 20 microM Dynasore with or without 30 microM Nocodazole. [00127] Surface Biotinylation of CaVl.2. After treatment, the cells were quickly washed and incubated with ice-cold lmg/ml EZ-linkTM Sulfo-NHS-SS-Biotin (Pierce) for 25 min. After 2 x 5min quenching of unbound biotin with 100 microM glycine, cells were washed and lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 2 niM NaF, 200 microM Na3VO4) supplemented with Complete Mini protease inhibitor cocktail (Roche). Total protein concentrations were determined and normalized between samples. The lysates were then incubated with prewashed NeutrAvidin coated beads at 4°C overnight. After washes, bound surface proteins were eluted and denatured, separated on NuPage gels (Invitrogen), and probed with rabbit anti-CaVl.2 antibody (Alomone).


Source: WO/2010/124240