I've been trying to isolation brain endothelial cells from mouse brain. Protocol that I've tweaked from one I found on bioprotocol:
- Cull via cervical dislocation, remove brain and put in ice cold DMEM/F12 (pre oxygenated for 15 mins). I usually remove 6 brains one after the other - takes around 10 mins.
- Remove cerebellum, olfactory bulbs, meninges, large vessels
- Homogenise brains (one by one) in glass homogeniser in DMEM/F12 supplemented with L-glut (2mM) and p/s (1%)
- Spin homogenate at 1350g for 5 min (4c)
- Resuspend pellet in 15mL 18% dextran solution supplemented with L-glut and p/s at 6000g for 10min (4c)
- Remove myelin layer and dextran, resuspend pellet in digestion media which contains DMEM/F12, collagenase (1mg/ml), DNAse (4ug/ml) and TLCK (0.147 ug/ml)
- Digest pellet for 1 hr 15 min at 37c, inverting tube every 15 min
- Spin at 1350g for 5 min RT
- Resuspend pellet in PBS
- Spin at 1350 for 5 min RT
- Resuspend in full media which contains DMEM/F12, 10% FBS, ECGS, anti-anti, L-glut, heparin
- Seed cells onto 6-well plate. 6 brains/6-well plate.
I've tried coating plates with rat tail collagen, and also gelatin, and have tried not coating the plates as well.
I change the media 24 hours later, and there is a lot of cell death Some of the vascular fraction sticks down, but not enough for the cells to be happy and surrounded by one another, so they end up not growing well at all :(
Please can someone help? I've wasted so many mouse brains trying to optimise this protocol :(
When we grow primary human microvascular endothelial cells, we are using endothelial cell growth medium and a bulletkit which are from Lonza.
Here is the link. Hope this would help. Good luck!
https://www.lonza.com/products-services/bio-research/primary-cells/human-cells-and-media/endothelial-cells-and-media/endothelial-cell-growth-media-kits.aspx
Hi Bethany,
We had great success in isolating mouse CVEC by using anti-PECAM-1 antibody and Dynal-450 magnetic bead approach (Handa O et al, Am J Physiol Heart Circ Physiol. 2008 Oct;295(4):H1712-9.). Nowadays you could use magnetic nanoparticle approach (Miltenyi) instead of Dynal one.
Important to note that you should try not to overexpose cells to collagenase treatment, that may reduce cell viability! In this regard you should also check enzymatic activity of collagenase that you use for tissue digestion, since collagenase from different source(s)/suppliers have different activity per weight unit. I would recommend no more than 500U/ml of collagenase II and 0.6U/ml dispase for brain tissue digestion.
In addition, fibronectin may be an alternative adhesive molecule to use instead of collagen, which has to be dissolved in acid for cell culture dish coating. If not properly washed, "collagen-coated" surfaces may acidify cell culture medium resulting in "very unhappy cells"....
Finally, you may check what MW of dextrans (which have extremely high MW range and different properties) is recommended for cell centrifugation step.
Best of luck.
Gedas
Bethany, we isolate cortical mouse brain endothelial cells with a similar protocol. Im happy to help and/or share the protocol. Send me a private or message or email, then we can discuss your problems!
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