Dear colleagues,
I've been trying to do proliferation assays using HUVECs and at the beginning we were using 20 ng/mL of VEGF as it was recommended by a colleague. However, this colleague uses this growth factor as positive control for proliferation because she studies its signaling. In my case, I just want to see if the conditions that I'm using are better or worse than the positive control.
The problem is that I have worse results when adding something "good" to HUVECs than in my negative control and I can't grisp what is going on. So VEGF didn't work and I changed for EGM-2 (so the EBM media with bulletkit for Lonza) as it is rich in several growth factors, but it also didn't work. I keep having better proliferation rates at the cells in starvation (no growth factors, only 1% antibiotics) than in my (supposedly) positive control.
Does any of you have a suggestion? And if you know, could you explain to me why is this happening (I think it goes against logic)?
Have you tried coating the dishes that you have seeded them? They may like collagen.
You have to cote the dish or flask with collagen. this type of cells have very slow proliferation rate and it easy to detached, I suggest to add more L-glutamine to the media.
One of the studies is regarding different coating surfaces but I've been getting non conclusive data, because the positive control works worse than no added factors. However, it is true that we had troubles with cell attachment lately.
We've tried using fibronectin and they don't seem to dettach, but same problem: positive control is not acting as positive control ...
Raheem Alabedi, I will take a look into L-glutamine in HUVECs :)
Thank you for your suggestions!
Dear Harald,
Yes, we did that but we kept having the same problem. We just started doing the same assay but in 24 well plates (as opposed to 96) and it looks like they're doing better. Apparently these cells aren't good option to be tested in 96 for reproducibility.
Thank you for helping out!
Best,
Cláudia
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