Asked 2nd Dec, 2017
Why are my HUVECs dying?
I am working with primary HUVEC cultures from Lonza. They are stored in liquid nitrogen at P5 in a freezing media of 90% FBS and 10% DMSO. They are plated on TC dishes coated with fibronectin in VascuLife EC media with VEGF at a density that has always been successful in the past. Media is changed the day after plating, and then every other day thereafter.
Sometimes the cells are able to proliferate and form a monolayer normally. However, in the past month, many of them experience death in large chunks of the plate starting the second day after plating (so after the first media change). For example, in a 6 well plate, one well may proliferate normally, two may experience almost complete cell death, and three will look normal in >50% of the well, but gone in the rest.
What is really confusing me is that they "balloon" out before death, a process that I am not familiar with. This occurs in cells handled by all lab members, so it is not one individual's technique in particular.
I have attached a 20x image showing a small area of healthy cells next to the sick ones.
Any guidance on how to remedy this issue would be greatly appreciated.
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