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Chitin-challenged Mice Model to Study M2 Macrophages Polarization   

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Original research article

A brief version of this protocol appeared in:
The Plant Cell
Jan 2015

Abstract

Chitin is a key component of insects, fungi, and house-dust mites. Chitin has been shown to induce M2-type immune responses in vivo. Intranasal or intraperitoneal (i.p.) administration of chitin particles results in infiltration of eosinophils to the local sites and activation of macrophages with a M2 phenotype. Chitin-challenged mice model can be used to induce M2 macrophages polarization and thus to analyze the M2 phenotype from isolated peritoneal cells.

Keywords: Chitin, Macrophages, M2 polarization, Mice

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.
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Jiménez-García, L., Herránz, S., Luque, A. and Hortelano, S. (2015). Chitin-challenged Mice Model to Study M2 Macrophages Polarization. Bio-protocol 5(17): e1584. DOI: 10.21769/BioProtoc.1584.
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