Ab10214

For culture, isolated cardiac cells were seeded at a density of 20,000 cells/mL on a 20 μg/mL laminin-pretreated support (Sigma), and cultivated in DMEM/F-12 supplemented with 10% FBS, 1 U/mL Na-Penicillin G, and 0.5 U/mL streptomycin (Gibco, Shanghai, China). In order to inhibit fibroblast proliferation, cultures were performed with 5 μg/ml cytosine □-D-arabinofuranoside (Ara C). Cells were incubated at 37 °C in a humidified, 5% CO₂–enriched atmosphere. These cells were cultured for four days. To visualize the proliferation of isolated cardiomyocytes, we fixed the cells in 4% paraformaldehyde for ten minutes. After washing, cells were permeabilized with 5% Triton-100 for 15 minutes. The cells were incubated with mouse monoclonal antibody against cardiac troponin T (Abcam, ab10214, 1:200 dilution) and rabbit anti–Ki67 antibody (Abcam, ab15580, 1:200 dilution) at 37 °C for two hours. After washing, cells were incubated with Alexa Fluor 555–conjugated anti–mouse second antibody (Abcam, ab150107, 1:1000 dilution) and Alexa Fluor 488–conjugated anti–rabbit second antibody (Abcam, ab150073, 1:1000 dilution) for 30 minutes. Nuclei were stained with DAPI. For quantification, ten different field pictures were taken from each well, and analyzed using Image J.

hPSC-derived cardiomyocytes were seeded on Matrigel-coated 20 mm coverslips in 12-well plates. Cells were washed with 1× PBS and fixed with 4% formaldehyde in PBS at room temperature for 10 min. The coverslips were washed and blocked with blocking buffer (1× PBS + 0.3% TritonX + 3% goat serum) at room temperature for 30 min. For staining, the following antibodies and concentration were used: anti-cTnT (ab10214, Abcam) 1:400, anti-SAA (ab9465, Abcam) 1:200, anti-Mlc2a (311 011, Synaptic Systems) 1:200 or anti-γ H2AX phosphoS139 (ab2893, Abcam). Antibodies were diluted in 1× PBS + 0.3% TritonX + 2% goat serum and incubated at 4 °C overnight. The cells were then washed three times for 5 min and stained with secondary antibodies (Alexa Fluor^® 488 or 647, Invitrogen) at a dilution of 1:200. The cells were stained with DAPI for 10 min in 1× PBS and after two more washes mounted onto microscope slides using ProLong^® Gold Antifade Mountant (Life Technologies).

Whole tissue lysates were prepared to determine troponin protein levels. Therefore, pulverized frozen tissue was homogenized in 40 μl/mg tissue 1× reducing sample buffer (106 mM Tris-HCl, 141 mM Tris-base, 2% lithium dodecyl sulfate (LDS), 10% glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.18 mM Phenol Red, 100 mM DTT) using a glass tissue grinder. Proteins were denatured by heating to 99 °C for 5 min and debris was removed by centrifugation at maximum speed for 10 min in a microcentrifuge (Sigma, 1–15 K).
For analysis of troponin protein levels by Western blot, 5 μg of protein were separated on a 4–15% TGX gradient-gel (Biorad) and transferred to a polyvinylidene difluoride membrane. Site-specific antibodies directed to cTnT (ab10214, Abcam), cTnT (T6277, Sigma-Aldrich), cTnT (ab8295, Abcam), cTnI (ab10231, Abcam), cardiac Troponin C (cTnC, sc48347, Santa Cruz) and α-actinin (A7811, Sigma-Aldrich) were used to detect the proteins which were visualized with an enhanced chemiluminescence detection kit (Amersham) and scanned with Amersham Imager 600. Protein levels were determined by densitometric analysis. Protein levels were normalized to α-actinin or cTnI when appropriate.
Equal loading of troponin complexes was verified with Imperial protein stain (Thermo Scientific).

Cells were harvested and rinsed three times with cold PBS and then fixed with 0.5% paraformaldehyde (PFA) for 20 min and permeabilized with 0.1% Triton X-100. Then, the cells were stained with anti-cTnT (1 : 500, ab10214, Abcam) followed by an Alexa Fluor 594 conjugated secondary antibody. cTnT positive cells were measured using a FACSCalibur flow cytometer (BD Bioscience, CA, USA).