Alexafluor 488 cojugated anti mouse igg

For cardiomyocyte characterization, immunostaining was performed using anti-cTNT (Abcam; 1:100) and anti-alpha-actinin (Sigma-Aldrich; 1:200) as primary antibodies and Alexafluor-488-cojugated anti-mouse IgG as a secondary antibody. Briefly, cells were washed with PBS (Life technologies), fixed with 4 % PFA (Sigma-Aldrich) for 15 min and permeabilized in PBS containing 0.1 % Triton X-100 (Sigma-Aldrich) and 5 % FCS (Life techologies) for 30 min. Cells were then incubated overnight with the primary antibodies. Next day, secondary antibody Alexafluor-488-cojugated anti-mouse IgG (1:1,000; Life technologies) were used to detect and visualize the primary antibodies. All antibodies were diluted in blocking solution. Similar protocol was also used for the staining using ISL1 (Biorbyt; 1:200) antibody. Micrographs were taken with an Axiovert 200 M microscope (Carl Zeiss).

1 × 10⁶ cells were trypsinized and fixed with 4 % PFA for 10 min. Cells were then washed with PBS, permeabilized in PBS containing 0.1 % Triton X-100 and 5 % FCS for 30 min and incubated for 2 h with cTNT antibody (Abcam; 1:100). No antibody was taken as a negative control. Cells were then washed once with PBS containing 0.1 % Tween-20 and resuspended in PBS containing 0.1 % Triton-X 100 and 5 % FCS and secondary antibody Alexafluor-488-cojugated anti-mouse IgG (1:1,000; Life technologies) for 1 h in dark. Finally, cells were washed again with PBS containing 0.1 % Tween-20 and measured for FACS analysis. Analysis was performed by Flow Jo program.