I have been using a 40% Dextran gradient centrifugation protocol with HBSS to isolate mouse capillaries (from cortical homogenate) but have not found any of my pericyte or smooth muscle cell markers in my vascular fraction (or at least not to any where near as high a yield as in my parenchymal fraction). I would appreciate any insight/help. Thanks!
Please check this paper.
https://www.ncbi.nlm.nih.gov/pubmed/11998866
I think that final concentration of dextran is 16% in my old protocol.
(4,500g 15 min) good luck!
I use magnetic bead sorting to isolate CD146+ cells from the lung...this includes both pericytes and endothelial cells. I then enrich for either of the two cell types using cell-specific growth medium.
Here's a methods paper we published a while ago, which should be helpful. There were followup papers, which you can also find. Good luck!
Nayak RC, Herman IM. Bovine retinal microvascular pericytes: isolation, propagation, and identification. Methods Mol Med. 2001;46:247-63. doi: 10.1385/1-59259-143-4:247.
Dear Jill,
Would you be able to forward me a protocol? Thanks!
Hi Cillian
Now, I am participating in Japanese viral meeting.
If I back to my home town, I will send our protocol.
Hi Cillian,
What in my case I do homogenize the whole brain in 3 mL PBS/FBS-5%, I centrifuge without dextran (1100g - 10 min - 4°C), discarding each time the supernatant until it gets translucent (3 rounds) and then I filtrate the resuspended pellet through a 100um mesh. Here I collect the filter-bound material that I centrifuge once again (same speed) and the pellet yields intact vessels with perycites when I check on a cavity slide with bright field microscope.
Did you manage to keep intact the brain microvessels apart from the parenchymal cells using Dextran ?
Which pericyte or smooth muscle cell markers are you using for which you have a positive signal for in your parenchymal fraction but not in the vascular fraction?
Also, are you doing a western blot or immunostaining? If so, how much protein yield do you get from one brain?
Dear Stephanie,
Thanks for the reply. I homogenize a whole cortex (without hipp, cerebellum, or brain stem) in 4.5mL HBSS, then add 4.5mL 40% dextran, mix, centrifuge at 6000g at 4C for 10 minutes. Then pour off my parenchyma, wash my vascular pellet, resuspend and wash again. The parenchyma is then made up to 15mL with HBSS, centrifuged again at 6000g for 10 minutes, soluble fraction taken off, and parenchyma pellet washed and resuspended.
I use PDGFRB as a pericyte marker and find a strong signal in both parenchyma and vascular fractions (but I believe I should only be seeing it in my vascular - the literature suggests it is greatest there). NG2 is also found in both fractions. Alpha smooth muscle actin is strongest in the vascular fraction. I cannot comment on protein yield at this time as I am still optimizing, however, it is usually only 1% or 2% that of the parenchyma.
I am doing western blots, however, what you have sugested regards viewing under the confocal is a good iea and I have been meaning to try this.
Question. You mention above that you filter through 100um (I wuld really appreciate the protocol for this if you could please share). Surely the filter-bound vessels would NOT be capillaries, and instead be larger arterioles and venules (as only the larger than 100um segments would stay on top)?
Hey Cillian,
Here's my email: [email protected] (beware, there is no letter i in my first name, hehe), I'll be glad to share with you our protocol which is still in optimization.
By the way I opened a thread where you can have a better idea of my protocol. The link is attached to this post.
About the type of vessels I can collect with a 100um mesh I came to the conclusion that all types of vessels are collected on the filter mesh since I don't believe that the pore size can discriminate the vessels by their size. This is supported by the different vessel sizes I can visualize with a classic microscope and my PI confirmed some of them were very likely to be capillaries. Now I need to get the corresponding signals...
I imagine the filter does the same as a collander would do with pasta: it would block most of them and some of them would go through as well as the liquid part.However, if you reduce the pore size of the mesh you most likely will be able to collect smaller vessels (like capillaries) but you also increase the chance to form a plug and block more of the debris you want to get rid of. But you could still do more rounds of filtration, which I intend to try very soon.
Also, I wonder if when you say that you wash your pellet I can assume that you are talking about another centrifugation step?
https://www.researchgate.net/post/How_can_I_separate_mouse_brain_vessels_from_parenchymal_cells_and_assess_purity_of_the_fractions_using_immunoblotting_western_blot_dot_blot?_tpcectx=profile_questions
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