Sep 30, 2020
Mouse Liver Single-Cell Dissociation with Cold Protease
- 1[Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States];
- 2[Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States;
- 3Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States]
Protocol Citation: Sanjay Subramanian, Stacey S Huppert 2020. Mouse Liver Single-Cell Dissociation with Cold Protease. protocols.io https://dx.doi.org/10.17504/protocols.io.bhpcj5iw
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: June 18, 2020
Last Modified: September 30, 2020
Protocol Integer ID: 38340
Abstract
This protocol is used to dissociate murine liver cells at 4 °C in an effort to preserve transcriptomic expression profiles in cells to be used for single-cell RNA sequencing and single-cell ATAC sequencing. An expedited protocol length limits the time of exposure to dissociative solutions in order to retain natural cellular distribution and viability while limiting transcriptomic changes arising from injury response, especially in fragile cell populations. This protocol has been tested and works in mice aged 2-4 weeks, dissociating cells from the left lobe for single-cell RNA sequencing using the 10x Chromium system.
This project was supported in part by NIH P30 DK078392 (Gene Analysis Core) of the Digestive Diseases Research Core Center in Cincinnati, R01DK107553, and the Cincinnati Pediatric Cell Atlas Center funded by Cincinnati Children’s Research Foundation to Stacey S. Huppert
Guidelines
Bacillus licheniformis enzyme mix
The Bacillus licheniformis mix is made up in DPBS at a concentration of 100mg/mL and stored at -80 °C in 100μL aliquots
To make 10mL of 1mg/mL Bacillus licheniformis mix:
9900μL DPBS
100μL Bacillus licheniformis 100mg/mL mix
10x PBS (1L)
80g NaCl
2g KCl
11.5g Na2HPO4 7H2O
2g KH2PO4
1L diWater
1x PBS for Flush
Make up 5 mL of 1x PBS by diluting 500μL 10x PBS in 4.5mL MilliQ water
Required Equipment
Refrigerated centrifuge
Tube rotator (or similar device)
Materials
MATERIALS
Protease from Bacillus Licheniformis SigmaCatalog #P5380
3 mL syringesBD BiosciencesCatalog #BD #309657
Falcon™ Cell Strainers, Mesh size: 70um; whiteThermo FisherCatalog #087712
William's E Medium, no glutamineThermo FisherCatalog #12551032
DPBS, calcium, magnesiumThermo FisherCatalog #14040133
100um Cell StrainerFisher ScientificCatalog #22363549
EqualFetal Bovine SerumAtlas BiologicalsCatalog #EF-0500-A
PrecisionGlide Needle 23GBD BiosciencesCatalog #BD #305120
BD Intramedic™ Polyethylene Tubing PE-50Fisher ScientificCatalog #BD #427516
Before start
Prepare essential solutions and leave on ice
Cool centrifuges to 4 °C
To make cannulation apparatus:
- Cut ~12 inch length of PE-50 tubing and make an angled cut at one end with a razor blade to serve as the cannulation tip.
- Carefully insert 23-gauge needle tip into opposite end of tubing, making sure not to poke a hole in tubing
- If a smaller cannula is needed to fit into the portal vein, the PE-50 can be warmed by rolling between fingers and then stretched by pulling. Additionally, PE-10 can be used for very small vessels.
Tissue Isolation
Tissue Isolation
20m
20m
Anesthetize mouse
Fill syringe with 1x PBS and twist on to needle with attached cannulation apparatus. Flick syringe and push plunger to ensure 1x PBS has filled the tubing and there are no air bubbles in the syringe or tubing
Open abdominal cavity and locate main trunk of portal vein. Make a small incision to allow for insertion of angled tip of cannulation apparatus without cutting all the way through portal vein
Make incision in inferior vena cava to allow blood and perfusing solutions to flow out
Cannulate the portal vein and flush 3-5mL ice cold 1x PBS through portal vein until liver tissue is sufficiently cleared of blood
5 mL ice-cold 1x PBS
Switch PBS syringe out for one with Bacillus licheniformis mixture while keeping the portal vein cannulated. Ensure no bubbles are in the system and then perfuse 3-5mL ice cold 1mg/mL Bacillus licheniformis mixture through the portal vein
5 mL ice-cold Bacillus licheniformis enzyme mix
Dissect out desired liver tissue and mince thoroughly with razor blade in petri dish with an additional 1mL Bacillus licheniformis mix; start timer for 1 hour
1 mL ice-cold Bacillus licheniformis enzyme mix
Dissociation and Filtration
Dissociation and Filtration
1h 5m
1h 5m
Transfer tissue-enzyme slurry to 5mL capped tube and incubate, rotate, or rock at 4 °C until timer reaches 1 hour
01:00:00 at 4 °C
Mince slurry again with razor blade, making sure to separate or scrape larger chunks to release the maximum number of cells
Pass slurry through a 100μM filter into a 50mL conical tube
Bring volume in tube up to 10mL by slowly passing ice cold Williams' E media with 20% FBS through filter to dislodge any sticking cells and stop the enzyme activity
10 mL Williams'' E 20% FBS with slurry
Centrifugation and Resuspension
Centrifugation and Resuspension
15m
15m
Centrifuge cells at 250g for 5min at 4 °C
00:05:00 centrifuge 250g at 4 °C
Discard supernatant and resuspend pellet in 5mL Williams E with 10% FBS
5 mL resuspend Williams' E 10% FBS
Filter suspension through a 70μM filter into a new 50mL conical tube; rinse filter with an additional 5mL of media
5 mL Williams' E 10% FBS
Repeat steps 9-10
00:05:00 centrifuge 250g at 4 °C
5 mL resuspend Williams' E 10% FBS
Note
Final resuspension volume may be greater or less than stated and should be judged accordingly based on pellet size
Quality Control
Quality Control
10m
10m
Analyze cell morphology using hemocytometer and Trypan blue
Note
Viable hepatocytes tend to take up trace amounts of Trypan blue
Adjust cell concentration as needed (700-1200 cells/μL for 10x Chromium system)
1000 cells/μL