Hello,
Sorry to hear your differentiations are not working.
Would you mind providing me with some more information on the cell line you are using? In particular, are you using one of our lines and does the line you are using have good stem cell morphology? (see Pictures of Healthy vs Unhealthy hiPSC colonies for examples). Could you post an example of how your stem cells look if they do not look similar to the pictures we have provided?
Although we do see variations in cTnT+ levels between our lines and from differentiation experiments, <1% cTnT+ is extremely low for a well with beating clusters and below the range we see even in subpar differentiations at day 12 using this protocol. Have you validated your flow protocol for staining and detecting cTnT in cardiomyocytes?
We have a video on our website showing our differentiation setups (https://youtu.be/cCVUrKGRINs). We have found that the timing of the media changes is important. Have you been changing the media from Chiron to IWP and IWP to B27- as close to 48hrs as possible?
If so, because your cTnT+ is so low my first suspicion would be that potentially something is off with one of the reagents. We lot test our B27 minus insulin. We have found that lot to lot variations can lead to unsuccessful differentiations. Have you tested multiple B27 minus insulin lots in a controlled experiment?
Thanks,
Haseeb