Isolation of ILC2 from Mouse Liver

Materials and Reagents

1. Liver from a IL-33 or IL-25 treated mouse (e.g. C57BL/6 or Balb/c)
   
2. Fetal calf serum (FCS)
   
3. RPMI-1640 medium
   
4. Easycoll separation solution (density 1.124) (Biochrom, catalog number: L6145 )
   
5. Anti-mouse CD16/CD32 (Fc-Block) (eBioscience, catalog number: 14-0161 )
   
6. 0.5 M CaCl₂ solution
   
7. 0.2 M MgCl₂ solution
   
8. AutoMACS rinsing solution (Miltenyi Biotec)
   
9. Brilliant Violet 421 Streptavidin (BioLegend, catalog number: 405226 )
   
10. Antibodies
    1. FACS antibodies
       1. Anti-Sca-1 (clone D7, FITC conjugated) (eBioscience, catalog number: 11-5981 )
          
       2. Anti-KLRG1 (clone 2F1, APC conjugated) (eBioscience, catalog number: 17-5893 )
          
       3. Anti-ICOS (clone 7E.17G9, PE conjugated) (eBioscience, catalog number: 12-9942 )
    2. Anti-mouse lineage-antibodies biotin labelled
       1. Anti-CD3 (clone 145-2C11) (eBioscience, catalog number: 13-0031 )
          
       2. Anti-CD45R (clone RA3-6B2) (eBioscience, catalog number: 13-0452 )
          
       3. Anti-Ly-6G (clone RB6-8C5) (eBioscience, catalog number: 13-5931 )
          
       4. Anti-CD11b (clone M1/70) (eBioscience, catalog number: 13-0112 )
          
       5. Anti-NK1.1 (clone PK136) (eBioscience, catalog number: 13-5941 )
          
       6. Anti-Ter-119 (clone Ter-119) (eBioscience, catalog number: 13-5921 )
          
       7. Anti-SiglecF (clone ES22-10D8) (Miltenyi Biotec, catalog number: 130-101-861 )
       8. Anti-CD5 (clone 53-7.3) (Miltenyi Biotec, catalog number: 130-101-960 )
          
11. Krebs-Ringer-Buffer (KRB) (see Recipes)
    
12. Collagenase IV solution (Sigma-Aldrich, catalog number: C5138 ) (see Recipes)
    
13. DNase I solution (Roche Diagnostics, catalog number: 10104159001 ) (see Recipes)
    
14. 30% Biocoll (see Recipes)
    
15. 80% Biocoll (see Recipes)
    
16. FACS buffer (see Recipes)
    
17. ACK buffer (see Recipes)
    
18. PEB buffer (see Recipes)
    

Equipment

1. GentleMACS C tube (Miltenyi Biotec)
   
2. 6 well plates
   
3. GentleMACS dissociator (Miltenyi Biotec)
   
4. MACSmix tube rotator (Miltenyi Biotec)
   
5. Centrifuge (Thermo Fisher Scientific, model: Multifuge X1R )
   
6. Cell sorter (e.g. FACSaria, BD Biosciences)
   
7. 100 µm cell strainer (Corning, catalog numer: 08-771-19 )
   
8. Hemocytometer
   
9. 15 ml Falcon tubes
   
10. 50 ml Falcon tubes
    
11. FACS tubes
    

Procedure

1. Freshly prepare liver digestion solution by pipetting 4.4 ml Krebs-Ringer-Buffer (KRB), 25 µl DNase I solution, 500 µl Collagenase IV solution, 20 µl 0.5 M CaCl₂ and 50 µl 0.2 M MgCl₂ into a gentleMACS C tube. Warm up by keeping at 37 °C for 30 min. This solution ensures effective liver tissue digestion while proteolytic degradation of immune cell surface receptors is limited.
   
2. Sacrifice mouse by cervical dislocation and remove the liver from a mouse aseptically under the sterile bench and transfer the organ into a well of a sterile 6 well plate filled with 5 ml KRB buffer. If co-isolation of some ILC2 cells potentially present in the vasculature should be avoided include liver perfusion e.g. via the portal vein system using standard techniques described elsewhere.
   
3. Rinse the liver with KRB buffer and transfer it into the C tube containing pre-warmed liver digesting solution.
   
4. Dissociate the tissue by applying the C tube into the gentleMACS Dissociator and running the program m_liver-01.02.
   Note: One mouse liver can be proceeded per C tube, the program can be run 2-3 times till the tissue is fully homogenized.
   
5. Attach the C tube containing the homogenized tissue to the MACSmix Tube Rotator and incubate the sample at 37 °C for 30 min under continuous rotation.
   
6. Apply the tube to the gentleMACS Dissociator again and run the program m_liver-02.02.
   
7. Prepare a 50 ml tube and a 100 µm cell strainer by rinsing them with PEB buffer.
   
8. Apply the homogenized tissue from C tube on the strainer on the 50 ml tube and wash the strainer with additional 10 ml PEB buffer.
   
9. Discard the cell strainer and add further 20 ml PEB buffer to the tube. Centrifuge the sample at 20 x g at 4 °C for 4 min. This step removes unwanted hepatocytes from the liver cell suspension.
   
10. Carefully remove the supernatant from the sample and transfer it into a new 50 ml tube.
    
11. Wash the cells by adding 30 ml PEB buffer and centrifugation at 300 x g for 10 min at 4 °C.
    
12. Carefully discard the supernatant and resuspend the cell pellet in 1 ml of PEB buffer.
    
13. Apply 10 ml of ACK buffer on the suspension to remove red blood cells. Incubate the sample at room temperature for 5 min.
    
14. Wash the sample two times by adding 30 ml PEB buffer and centrifugation at 300 x g for 10 min at 4 °C.
    
15. Fill 15 ml tube with 5 ml of 80% Biocoll separation solution.
    
16. Resuspend isolated cells in 10 ml 30% Biocoll separation solution and overlay slowly to the 80% Biocoll solution. Centrifuge the cells at room temperature with 1,400 x g for 20 min without breaks.
    
17. Collect the target cells by carefully pipetting up the interface fraction between the gradients into a new 50 ml tube.
    
18. Wash with 30 ml PEB buffer and centrifuge at 300 x g for 15 min.
    
19. Count the cells by using a hemocytometer and distribute to FACS tube for staining.
    
20. Prior to staining block unspecific bindings by incubating cells with 1 µl/10⁷ cells of Fc-Block at 4 °C for 5 min.
    
21. Proceed immediately to staining using the following antibodies using the manufacturer’s recommended concentrations: anti-Sca-1, anti-ICOS, anti-CD25, biotinylated lineage antibodies. Add 0.015 µg/10⁶ cells of Brilliant Violet 421 Streptavidin.
    
22. Incubate the tube at room temperature for 20 min.
    
23. Wash the cells by adding 2 ml FACS buffer and centrifuge at 300 x g for 10 min.
    
24. Proceed to FACS sorting. Gate and sort ILC2 as lineage negative, Sca-1⁺, ICOS⁺, KLRG1⁺ cells (see gating strategy in Figure 1).
    
    
    Figure 1. Gating strategy
    

Recipes

Note: All materials and reagents should be low endotoxin cell culture quality.

1. Krebs-Ringer-Buffer (KRB)
   154 mM NaCl
   5.6 mM KCl
   5.5 mM glucose
   20.1 mM NaHCO₃
   Adjust pH to 7.4 with NaOH
   Bring to final volume of 1,000 ml
   Filter sterilize and store at 4 °C
   
2. Collagenase IV solution
   5,000 Digestion Units/ml in KRB buffer
   Store stock solution at -20 °C
   
3. DNase I solution
   30,000 Units/ml in KRB buffer
   Store stock solution at -20 °C
   
4. 30% Biocoll
   36.97 ml Biocoll 100%
   63.03 ml PBS
   Keep sterile at 4 °C
   
5. 80% Biocoll
   79.8 ml Biocoll 100%
   20.1 ml PBS
   Keep sterile at 4 °C
   
6. FACS buffer
   Add 5 ml fetal calf serum to 495 ml of PBS
   
7. ACK buffer
   4.145 g NH₄Cl
   0.5 g KHCO₃
   18.6 g EDTA
   500 ml distilled water
   Filter sterilize and keep at 4 °C
   
8. PEB buffer
   Phosphate buffered saline (PBS) containing 0.5 % BSA and 2 mM EDTA
   

Acknowledgments

This work was supported by the Collaborative Research Center 796 and the Priority program SPP1656 of the DFG (to S.W.) and the Interdisciplinary Center for Clinical Research (IZKF) of the University Medical Center Erlangen.

References

1. Mchedlidze, T., Waldner, M., Zopf, S., Walker, J., Rankin, A. L., Schuchmann, M., Voehringer, D., McKenzie, A. N., Neurath, M. F., Pflanz, S. and Wirtz, S. (2013). Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis. Immunity 39(2): 357-371.