I am working with primary HUVEC cultures from PromoCell. They proliferate well using ECGM media with 2% FBS from PromoCell (Ref. C-22010), and the cells look healthy and grow successfully. Then, we seed the cells in 96-well (LUMOX Sarstedt) and 6-well plates (Sarstedt) at 5,000 cells/well and 50,000 cells/well, respectively. After 24h, the cells appear healthy under the microscope. We then remove the media to add fresh media with our treatments. However, just after doing this, cells appear unhealthy in some, but not all, wells. In fact, we have observed this "damage" just in some areas of the well, most of the time. Initially, I thought that it was due to the treatment because it occurs more frequently in treated wells. However, I have also observed it in control wells. I wondered if this erratic behavior was due to mycoplasma contamination, but upon testing, there was no contamination by mycoplasma. This phenomenon is more pronounced when we use a 96-well plate compared to a 6-well plate.

This erratic behavior is really confusing.

Has anyone observed similar behavior with their cells? I would really appreciate any suggestions or advice on how to address this problem. Thanks in advance.

I have attached some images taken with a HoloMonitor.