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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.

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Chitin-challenged Mice Model to Study M2 Macrophages Polarization
基于几丁质刺激的小鼠模型研究M2巨噬细胞的极化

免疫学 > 免疫细胞功能 > 巨噬细胞
作者: Lidia Jiménez-García
Lidia Jiménez-GarcíaAffiliation: Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid, Spain
Bio-protocol author page: a2527
Sandra Herránz
Sandra HerránzAffiliation: Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid, Spain
Bio-protocol author page: a2528
Alfonso Luque
Alfonso LuqueAffiliation: Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos IIIInstituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid, Spain
Bio-protocol author page: a2529
 and Sonsoles Hortelano
Sonsoles HortelanoAffiliation: Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid, Spain
For correspondence: shortelano@isciii.es
Bio-protocol author page: a2530
Vol 5, Iss 17, 9/5/2015, 1889 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1584

[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(甲壳素), Macrophages(巨噬细胞), M2 polarization(M2极化), Mice(小鼠)

[Abstract]

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.

材料和试剂

  1. 小鼠C57BL/6雄性(年龄8-12周)
  2. 甲壳素(Sigma-Aldrich,目录号:C9752)
  3. 70%乙醇
  4. PBS(Lonza,目录号:BE17-515Q)
  5. 70μm无菌细胞过滤器(BD Biosciences,目录号:352350)
  6. RPMI 1640(Lonza,目录号:BE 12-115F)
  7. 10毫升注射器
  8. 21 G针
  9. 25 G针
  10. 15 ml锥形管

设备

  1. 超声波浴,P-selecta ultrason(150 W)
  2. 剪刀
  3. 台式冷冻离心机

程序

  1. 甲壳素施用
    1. 几丁质的制备
      1. 每只小鼠重量800ng,用PBS洗涤3次(5分钟,13,000rpm,16,800×g )。
      2. 将几丁质悬浮在1ml PBS中,并在浴中超声处理1小时[在超声器(Ultrason,Selecta)的最大设置下]。
        注意:或者,甲壳素可以使用Branson超声波仪在25%输出功率下超声处理3次,每次5分钟。
      3. 用70μm无菌细胞过滤器过滤悬浮液。
    2. 注射1ml几丁质悬浮液。 每只小鼠使用10毫升 注射器用25 G针。 等待2天,收获腹膜 细胞

  2. 分离几丁质诱发的腹膜渗出液细胞(PECs)
    1. 牺牲小鼠与CO 2或异氟烷(使用颈脱位   不在本协议中指示,以避免潜在的内部 出血)。
    2. 用70%乙醇清洁腹部。
    3. 去除皮肤暴露腹膜壁。 在皮肤上做一个小切口,并牢固地拉紧
    4. 用25 G针将10ml RPMI 1640注入腹膜腔

  3. 分离几丁质诱发的腹膜渗出液细胞(PECs)
    1. 牺牲小鼠与CO 2或异氟烷(使用颈脱位   不在本协议中指示,以避免潜在的内部 出血)。
    2. 用70%乙醇清洁腹部。
    3. 去除皮肤暴露腹膜壁。 在皮肤上做一个小切口,并牢固地拉紧
    4. 用25 G针将10ml RPMI 1640注入腹膜腔...
    5. 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)。 TSC-mTOR通路调节巨噬细胞极化。 Nat Commun 4:2834。
  2. Jiménez-García,L.,Herránz,S.,Luque,A。和Hortelano,S。(2015)。 p38 MAPK在IL-4诱导的腹膜巨噬细胞的替代活化中的关键作用。 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, Honma,K.,Matsuyama,T.,Yui,K.,Tsujimura,T.,Standley,DM,Nakanishi,K.,Nakai,K.and Akira,S。(2010)。 Jmjd3-Irf4轴调节M2巨噬细胞极化和宿主对抗蠕虫感染的反应。 Nat Immunol 11(10):936-944。
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How to cite this protocol: 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; Full Text



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