I need help with primary mouse brain endothelial cell isolation and cultivation. My cells do not attach to the tissue culture plate (coated with fibronection) after overnight incubation. They are not stained by Trypan blue prior to seeding.
I used ~6 months old mice following the Miltenyi protocol (https://www.miltenyibiotec.com/US-en/applications/all-protocols/isolation-and-cultivation-of-endothelial-cells-from-adult-mouse-brain.html) with some modifications:
1. Instead of using adult brain dissocation kit (Cat# 130-107-677), I used neural dissocation kit (Cat# 130-092-628).
2. For all the centrifugation, I used 400g x 7 min, instead of 300g x 10 min.
3. I didn't perform RBC lysis. Instead, I performed transcardial perfusion before dissecting the brain.
I also have a question regarding CD45. The protocol suggest removal of CD45+ cells with negative selection. Is that truly necessary?
Thanks!
