Chitin-challenged Mice Model to Study M2 Macrophages Polarization

Materials and Reagents

1. Mice C57BL/6 male (age 8-12 weeks)
   
2. Chitin (Sigma-Aldrich, catalog number: C9752 )
   
3. 70% ethanol
   
4. PBS (Lonza, catalog number: BE 17-515Q )
   
5. 70 μm sterile cell strainers (BD Biosciences, catalog number: 352350 )
   
6. RPMI 1640 (Lonza, catalog number: BE 12-115F )
   
7. 10 ml syringe
   
8. 21 G Needles
   
9. 25 G Needles
   
10. 15 ml conical tubes
    

Equipment

1. Sonication bath, P-selecta ultrasons (150 W)
   
2. Scissors
   
3. Bench-top refrigerated centrifuge
   

Procedure

1. Chitin administration
   
   1. Preparation of chitin
      
      1. Weight 800 ng of chitin per mouse and wash 3 times with PBS (5 min, 13,000 rpm, 16,800 x g).
         
      2. Suspend chitin in 1 ml of PBS and sonicate in bath for 1 h [at the maximum setting of the sonicator (Ultrasons, Selecta)].
         Note: Alternatively chitin can be sonicated at 25% output power three times for 5 min with a Branson sonicator.
         
      3. Filter the suspension with 70 μm sterile cell strainers.
   2. Inject 1 ml of chitin suspension i.p. per mouse using a 10 ml syringe with a 25 G needle. Wait for 2 days and harvest peritoneal cells.
      
   
   
2. Isolation of chitin elicited peritoneal exudate cells (PECs)
   
   1. Sacrifice mouse with CO₂ or isofluorane (the use of cervical dislocation is not indicated in this protocol in order to avoid potential internal bleeding).
      
   2. Clean abdomen with 70% ethanol.
      
   3. Remove skin to expose the peritoneal wall. Practice a small incision in the skin and pull firmly.
      
   4. Inject 10 ml of RPMI 1640 into the peritoneal cavity with a 25 G needle.
      
   5. Massage abdomen for approximately 30 sec.
      
   6. Recover peritoneal fluid as much as possible using a 10 ml syringe with a 21G needle. (Usually approximately 8-10 ml fluid can be recovered from one mouse.)
      
   7. Remove needle from syringe and put fluid into a 15 ml conical centrifuge tube on ice.
      
   8. Centrifuge peritoneal cells 5 min at 300 x g (1,500 rpm, 4 °C). Discard the supernatant and collect the pellet.
      
   9. Resuspend cell pellet in 10 ml of RPMI 1640 medium and count cells. (The cell number expected to be recovered is about 5-8 millions.)
      
   10. Analyze peritoneal cells to confirm M2 activation by performing either real-time PCR or flow cytometry techniques to detect typical M2 markers (e.g. Arginase-1, Ym-1, Fizz-1, mannose receptor).

Acknowledgments

This study was supported by grant PI11.0036 from the FIS and MPY 1410/09 from ISCIII to SH, and by grant TPY-M-1068/13 to AL. AL was supported by MINECO-ISCIII, through the Miguel Servet Programme (CP12/03087). L. J-G. was supported by FIS (FI12/00340). This protocol is adapted from Jimenez-Garcia el at. (2015).

References

1. Byles, V., Covarrubias, A. J., Ben-Sahra, I., Lamming, D. W., Sabatini, D. M., Manning, B. D. and Horng, T. (2013). The TSC-mTOR pathway regulates macrophage polarization. Nat Commun 4: 2834.
   
2. Jiménez-García, L., Herránz, S., Luque, A. and Hortelano, S. (2015). Critical role of p38 MAPK in IL-4-induced alternative activation of peritoneal macrophages. Eur J Immunol 45(1): 273-286.
   
3. Satoh, T., Takeuchi, O., Vandenbon, A., Yasuda, K., Tanaka, Y., Kumagai, Y., Miyake, T., Matsushita, K., Okazaki, T., Saitoh, T., Honma, K., Matsuyama, T., Yui, K., Tsujimura, T., Standley, D. M., Nakanishi, K., Nakai, K. and Akira, S. (2010). The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat Immunol 11(10): 936-944.