3434-010-02) and N21-MAX Insulin Free Media Supplement (
AR010) along with Recombinant Human Activin A on day 0, Recombinant Human FGF-basic (
4114-TC), Recombinant Human BMP-4, and CHIR99021 (
4423) on days 1-5, and Recombinant Human Dkk-1 (Catalog # 5439-DK) on days 5-7 to induce cardiomyocyte differentiation. Cells were then cultured in media supplemented with N21-MAX Insulin Free Media Supplement (
AR010) on days 7-12 and media supplemented with N21-MAX Media Supplement (
AR008) from day 12 and beyond. Cells were fixed and stained for stage-specific markers at select time points during the procedure. The pluripotency marker Oct-4 was detected using a Mouse Anti-Human Oct-4A Monoclonal Antibody (
MAB17591). The mesoderm marker, Snail is expressed intermediately during differentiation (Day 1). It was detected using a Goat Anti-Human Snail Polyclonal Antibody (
AF3639). The cardiomyocyte markers NKX2.5 and Troponin T are not present in cells during early (Day 0) and intermediate (Day1) differentiation and become more highly expressed during the later stages of differentiation (Day 7, Day 30). NKX2.5 was detected using a Goat Anti-Human NKX2.5 Polyclonal Antibody (
AF2444) and Troponin T was detected using a Mouse Anti-Human Cardiac Troponin T Monoclonal Antibody (
MAB1874). Snail and NKX2.5 primary antibodies were visualized with a NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (
NL001). Oct-4 and Troponin T were visualized with a NorthernLights 557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (
NL007)." class="big_lightbox" target="_blank">
3434-010-02) and N21-MAX Insulin Free Media Supplement (
AR010) along with Recombinant Human Activin A on day 0, Recombinant Human FGF-basic (
4114-TC), Recombinant Human BMP-4, and CHIR99021 (
4423) on days 1-5, and Recombinant Human Dkk-1 (Catalog # 5439-DK) on days 5-7. Following culture in media supplemented with N21-MAX Insulin Free Media Supplement (
AR010) on days 7-12 and media supplemented with N21-MAX Media Supplement (
AR008) from day 12 and beyond, cells were fixed and stained for the atrial-specific marker, MLC2a, and the ventricle-specific marker, MYH7 using a Rabbit Anti-Human MYH7 Monoclonal Antibody (
MAB90961)." class="big_lightbox" target="_blank">