We are interested in isolating and culturing rat lung microvascular endothelial cells. Any help is highly appreciated.
@Abha Sahni: Thank you for the protocol. I am looking for protocol for isolating ECs from lungs. Do you use the same protocol for lungs as well? If so, how much pure population you get and how long (passages) we can keep them in culture? Our isolations failed so far due to the overgrowth of fibroblast-like cells. Thanks
Characterization of microvascular endothelial cells isolated from the dermis of adult
mouse tails Microvascular Research 82 (2011) 97–104
Nicely describes magnetic activated cell sorting (MACS) and further culture applicable to ECs of any origin.
@Abha Sahni. I have few questions about their microvascular endothelial cell isolation protocol in the publication that you provided, and I would appreciate if you can help with below questions.
In the protocol it says 'finely mince' the tissue. How big are the chunks should be (approx.) and how long do you mince?. Do you use which coating (gelatin, fibronectin, matrigel) when you plate the cells? Do they have cobblestone EC morphology after 5 passages? Do you pool lungs to get enough cells? Do the PECAM -coated Dyna beads are good for RAT ECs and if so which provider? How much purity (%) of cells you get?
Sorry for many questions as we are facing many problems for almost an year. Thanks for your help.
Sorry for the late reply. We mince the tissue using the scissors for at least 5 min. (keep on ice) as smaller chunks as possible. We use 0.2% gelatin to coat the surface. Yes, if the cells population is pure you will see the correct morphology. We don't pool lungs, but you can. We more often isolate mice lung Ecs, but I am sure coated Dyna beads will work for the rat Ecs too. We buy it from Life Technologies and mostly get about 90-95% pure Ecs. Good Luck.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology