Figure 2
The impact of confluency on differentiation efficiency. The figure shows the morphology of Detroit551-A cell culture with different confluency prior to differentiation initiation and monitoring them at different stages of differentiation process. Finally estimation of TNNT2 + cells in the culture using flow cytometry. (A) Cells cultured to a confluency of 60–70% prior to differentiation initiation. Beating cardiomyocytes appeared on day 7 of differentiation and staining the cells for TNNT2 marker showed a high level of TNNT2 + cells on day 15. (B) This panel shows Detroit 551-A at a starting confluency of 80–90% on day 1 and the presence of few beating clumps on day 8 after initiation of differentiation. Flow cytometry revealed low level of TNNT2 + cells in the culture on day 15. (C) This panel shows morphology of Detroit 551-A cells, cultured to a confluency of 30–40% prior to differentiation. This initial confluency gave rise to no cardiomyocytes. Cells started to die just after 24 h treatment with CHIR99021 (day-1) and most were dead after 3 days. All Pictures were taken using inverted light microscope (Leica DML 1000). Scale bars, 500 μm. Cardiomyocyte population were estimated using flowcytometer Sony cell sorter SH800 (Sony Biotechnology Inc.) and data were analyzed and visualized using FlowJo 10.5.0 (FlowJo LLC, OR, USA) software (www.flowjo.com).
