TITLE: EC isolation from human mesenteric artery for scRNA-seq
AUTHORS: Xiaofang  Tang, Kiran Sriram, Yingjun Luo, Zhen Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines a method to efficiently isolate the human mesenteric arterial endothelial cells. Following the physical scraping, endothelial cells from the inner surface of the vessels are separately liberated using a digestion buffer solution. The procedure takes approximately one hour and has been validated for down-stream applications such as FACS, VE-cad immunostaining and single cell RNA profiling.</div></div>

[STEPS]
?. [EC isolation from human mesenteric artery for scRNA-seq]
Wash freshly isolated human mesenteric artery (~5 cm in length) with DPBS in a 10-cm petri-dish. Use sterile surgical tools to clean the vessel (Removing the fat and outer connective tissues).
?. [EC isolation from human mesenteric artery for scRNA-seq]
Cut open the vessel and expose the inner lumen. Anchor the four corners of the vessel using 23G needles on a black wax.
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Add  to the lumen side of the artery.
[TrypLE]
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Transfer  to a 5 mL tube.
[cell suspension]
?. [EC isolation from human mesenteric artery for scRNA-seq]
Add equal volume () of M199 medium (or any EC growth media) to stop enzyme digestion.
1 mL
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Remove the supernatant and re-suspend the pellet gently and thoroughly with  using wide-orifice LiteTouch pipette tips.
[0.04% BSA-DPBS]
?. [EC isolation from human mesenteric artery for scRNA-seq]
Pass the cell suspension through a 40 μm strainer to remove any cell debris. Wash the strainer by adding another .
[0.04% BSA-DPBS]
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Remove the supernatant and re-suspend the pellet with  by using wide-orifice LiteTouch pipette tips and gently pipetting up and down along the inner wall of the tubes.
[0.04% BSA-DPBS]
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Count the cell viability: Mix  + . Take  to count cell number.
[trypan blue]
[cell suspension]
[mixture]
Ensure there's no cell debris, significant number of doublets or cell clumps in the suspension. Always keep the cells  .
Usually we could get at ~ 105 cells in total.
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Use appropriate number of cells for scRNA library preparation following the 10XGenomics 3' Expression protocol. The rest of the cells can be cultured or used for other downstream assays.
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Centrifuge at   for .
Centrifuge: 600 34, 4 10
?. [EC isolation from human mesenteric artery for scRNA-seq]
The EC isolation results.
?. [EC isolation from human mesenteric artery for scRNA-seq]
Use a scalpel to scrape the lumen 10-15 times (the purpose is to dissociate the endothelium of the intima).
?. [EC isolation from human mesenteric artery for scRNA-seq]
Keep the remaining vessel in DPBS . The rest of the vessel can be scraped again to collect the vascular smooth muscle cells in the intima layer.
on ice
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Incubate with shaking at ~ for .
Centrifuge: 150 33, 37 10
?. [EC isolation from human mesenteric artery for scRNA-seq]
Centrifuge at  for .
Centrifuge: 600 34, 4 10