Sep 26, 2020
EC isolation from human mesenteric artery for scRNA-seq
This protocol is a draft, published without a DOI.
- 1City of Hope
Protocol Citation: Xiaofang Tang, Kiran KS Sriram, Yingjun Luo, Zhen Chen 2020. EC isolation from human mesenteric artery for scRNA-seq. protocols.io https://protocols.io/view/ec-isolation-from-human-mesenteric-artery-for-scrn-bf37jqrn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 06, 2020
Last Modified: September 26, 2020
Protocol Integer ID: 36703
Keywords: endothelial cells, isolation, EC isolation, human, mesenteric artery, human mesenteric artery, scRNA-seq,
Abstract
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.
Guidelines
Cells should be washed and resuspended in LoBind 2.0 mL eppendorf tubes and wide-bore pipette tips for better cell viability.
Pipette the cell suspension very gently and slowly for all steps involved.
Each 4-5 cm vessel, use 1 mL TrypLE .
Keep the tissues/cells On ice .
Materials
MATERIALS
DPBS (10X), calcium, magnesiumThermo FisherCatalog #14080055
Medium 199Merck MilliporeSigma (Sigma-Aldrich)Catalog #M2520-10X
40 μm cell strainerFisher ScientificCatalog #14100150
Trypan Blue SolutionCorningCatalog #MT25900CI
Disposable Safety ScalpelsMyco InstrumentationCatalog #6008TR-10
Fisher BioReagents™ Bovine Serum Albumin Heat Shock TreatedFisher ScientificCatalog #BP1600-100
Pipette Tips RT LTS 1000µL LW 768A/8RaininCatalog #30389219
DNA LoBind Tube 2.0 mLEppendorfCatalog #022431048
Pipet-Lite Pipette Unv. SL-1000XLS RaininCatalog # 17014407
30 G X 1 in Needle Becton Dickinson (BD)Catalog #305128
Pipette Tips RT LTS 1000µL FLW 768A/8Mettler ToledoCatalog #30389218
TrypLE™ Express Enzyme (1X) phenol redThermo Fisher ScientificCatalog #12605010
STEP MATERIALS
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Additional materials required:
- 10-cm petri-dish
- 5 mL tubes
- Sterile surgical tools
- Candle platform
- 0,04% BSA-DPBS
Protocol materials
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Disposable Safety ScalpelsMyco InstrumentationCatalog #6008TR-10
DPBS (10X), calcium, magnesiumThermo FisherCatalog #14080055
Pipette Tips RT LTS 1000µL FLW 768A/8Mettler ToledoCatalog #30389218
Trypan Blue SolutionCorningCatalog #MT25900CI
Medium 199Merck MilliporeSigma (Sigma-Aldrich)Catalog #M2520-10X
TrypLE™ Express Enzyme (1X) phenol redThermo Fisher ScientificCatalog #12605010
Pipet-Lite Pipette Unv. SL-1000XLS RaininCatalog # 17014407
40 μm cell strainerFisher ScientificCatalog #14100150
Fisher BioReagents™ Bovine Serum Albumin Heat Shock TreatedFisher ScientificCatalog #BP1600-100
Pipette Tips RT LTS 1000µL LW 768A/8RaininCatalog #30389219
DNA LoBind Tube 2.0 mLEppendorfCatalog #022431048
30 G X 1 in Needle Becton Dickinson (BD)Catalog #305128
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Warm up 1 mL TrypLE at 37 °C for 00:05:00 before use.
Prepare at least 6 mL 0.04% BSA-DPBS .
EC isolation from human mesenteric artery for scRNA-seq
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).
Freshly isolated human mesenteric artery.
Cut open the vessel and expose the inner lumen. Anchor the four corners of the vessel using 23G needles on a black wax.
Anchored vessel.
Add 1 mL TrypLE to the lumen side of the artery.
Use a scalpel to scrape the lumen 10-15 times (the purpose is to dissociate the endothelium of the intima).
Keep the remaining vessel in DPBS On ice . The rest of the vessel can be scraped again to collect the vascular smooth muscle cells in the intima layer.
Transfer 1 mL cell suspension to a 5 mL tube.
Incubate with shaking at ~150 rpm, 37°C for 00:05:00 .
Add equal volume (1 mL )) of M199 medium (or any EC growth media) to stop enzyme digestion.
Centrifuge at 600 x g, 4°C for 00:05:00 ..
Remove the supernatant and re-suspend the pellet gently and thoroughly with 1 mL 0.04% BSA-DPBS using wide-orifice LiteTouch pipette tips.
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Pass the cell suspension through a 40 μm strainer to remove any cell debris. Wash the strainer by adding another 4 mL 0.04% BSA-DPBS .
Centrifuge at 600 x g, 4°C for 00:05:00 .
Remove the supernatant and re-suspend the pellet with 0.5 mL 0.04% BSA-DPBS by using wide-orifice LiteTouch pipette tips and gently pipetting up and down along the inner wall of the tubes.
Pipette Tips RT LTS 1000µL FLW 768A/8RaininCatalog #30389218
Count the cell viability:
Mix 10 µL trypan blue + 10 µL cell suspension . Take 10 µL mixture to count cell number.
Note
Ensure there's no cell debris, significant number of doublets or cell clumps in the suspension.
Always keep the cells On ice .
Expected result
Usually we could get at ~ 105 cells in total.
The EC isolation results.
Expected result
Morphology of isolated mesenteric arterial endothelial cells after two passages (Passages 2 and 3).
CD31 flow cytometry of isolated mesenteric arterial ECs, HUVEC was used as a positive control
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.