Day4:Cell Adhesion/Spreading/Growth (Trial 3)
After four days of this ongoing HUVEC culture in pncSi transwell, HUVECs look much better than before. Here I have freshly stained them with Calcein AM and we can see how bright it is in comparison to CMFDA stained cells (or may be there’s some flaw in my CMFDA staining procedure). Till now its been observed that the density on day2 is lower than on day0 (4-5 hrs after plating). This indicates, there are cells that detach from the surface and the upside-down format of the membrane could have promoted this. On day4, the spreading and cell density is higher than on day2, and cells look ~60% confluent.
As seen above, these cells are very close to normal morphology. There are few cells that look rounded/shrunk, which could be due to the 30-45 min incubation @ RT during live staining. Also, hanging from the surface might be responsible for cells to slowly detach. Further time point of this experiment could confirm whether all the cells attain normal morphology and proliferate or few of them continue to detach.
It’s strange that ~ 50% of the cells are rounded/clumped and ~50% of the cells exhibit normal morphology. It’s nice to see that at least some of the cells have normal morphology after 4 days.