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Protocol for Macrophage Depletion from Mice
清除小鼠巨噬细胞的方法   

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Abstract

Macrophage depletion has been used extensively to study autoimmune disease and more recently in tumor models. The clodronate-containing liposomes will be recognized as foreign particles and get engulfed by macrophages upon dosing into the animal by the chosen routes. Consequently, macrophages that have engulfed liposomes will all be destroyed by the liposomal. In the protocol presented here the clodronate-containing liposomes were used to systemically deplete macrophages in mice.

Materials and Reagents

  1. Mice
  2. Clodronate (dichloromethylene diphosphonic acid)-loaded liposomes (VUmc)
  3. PBS-containing liposomes (VUmc)
  4. Phosphatidyl choline (Lipoid GmbH, catalog number: D-67065)
  5. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  6. Liposomes (see Recipes)
  7. Liposomes suspended in sterile phosphate buffered saline (see Recipes)

Equipment

  1. Syringe (10 ml and 1 ml) (BD, catalog numbers: 309604 and 305111 )
  2. 26 G 1/2 needles (BD, catalog number: 309659 )

Procedure

  1. Twenty, 8-week-old mice were randomly divided into two groups.
  2. One group of mice were intravenously injected t using 1-ml syringe with 26 G 1/2 needles, twice per week with clodronate-loaded liposomes in 150 µl for the first month and 100 µl following the first month’s injection to reduce possible side effects, such as infection, live toxicity et al. The mice were treated for 3 months totally.
  3. The second group was treated with PBS-liposomes.
  4. At different time points (such as 2, 4, 8 weeks after treatments) of the experiment, one or two mice from each group will be euthanized to examine inflammation and other interesting analysis. Based on our experiments, the FACS analysis detected around 90% of macrophage reduction in the group that treated with the clodronate-loaded liposomes versus the control group after 2 weeks treatment, and this reduction kept to the end of the experiments.
  5. During the whole treatment period, the treated mice need to be closely monitored for the possible signs and symptoms such as weight loss, yellow, bloody, or tar-colored stool, dark urine color, diarrhea, fatigue etc.
  6. At the end of the experiment, (depend on the experiment), the mice were euthanized and the lung tissues were collected for various analyses.

Recipes

Notes:

  1. Based on the instructions from company.
  2. The clodronate-loaded liposomes and PBS-containing liposomes are ready for use, but the solutions that usually kept in 4 °C need to be warmed up to room temperature before injections.
  1. Liposomes
    Liposomes are artificial lipid spherules made of:
    1. Phosphatidyl choline
    2. Cholesterol
  2. Phosphate buffered saline (PBS)
    1. 10 mM Na2HPO4
    2. 10 mM NaH2PO4
    3. 140 mM NaCI

Acknowledgments

We thank Yinling Hu, Robert H. Wiltrout and Jami Willette-Brown from Frederick National Laboratory for Cancer Research for their support and suggestions on the experimental design. This work was supported by the National Cancer Institute (ZIA BC 011212 and ZIA BC 011391 to Y.H.).

References

  1. Xiao, Z., Jiang, Q., Willette-Brown, J., Xi, S., Zhu, F., Burkett, S., Back, T., Song, N. Y., Datla, M., Sun, Z., Goldszmid, R., Lin, F., Cohoon, T., Pike, K., Wu, X., Schrump, D. S., Wong, K. K., Young, H. A., Trinchieri, G., Wiltrout, R. H. and Hu, Y. (2013). The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas. Cancer Cell 23(4): 527-540.

简介

巨噬细胞缺失已广泛用于研究自身免疫疾病和最近在肿瘤模型中。 含有氯膦酸盐的脂质体将被认为是外来颗粒,并且在通过所选择的途径给药到动物中时被巨噬细胞吞噬。 因此,吞噬脂质体的巨噬细胞将全部被脂质体破坏。 在本文提出的协议中,含氯膦酸盐的脂质体用于系统性消耗小鼠中的巨噬细胞。

材料和试剂

  1. 小鼠
  2. 氯屈酸(二氯亚甲基二膦酸)负载的脂质体(VUmc)
  3. 含有PBS的脂质体(VUmc)
  4. 磷脂酰胆碱(Lipoid GmbH,目录号:D-67065)
  5. 胆固醇(Sigma-Aldrich,目录号:C8667)
  6. 脂质体(参见配方)
  7. 悬浮于无菌磷酸盐缓冲盐水中的脂质体(参见Recipes)

设备

  1. 注射器(10ml和1ml)(BD,目录号:309604和305111)
  2. 26 G 1/2针(BD,目录号:309659)

程序

  1. 将20只8周龄的小鼠随机分为两组。
  2. 一组小鼠使用1-ml注射器用26G 1/2针头静脉内注射,每周两次,负载氯膦酸盐的脂质体,第一个月为150μl,第一个月注射后为100μl,以减少可能的副作用,例如感染,活毒性等。小鼠完全治疗3个月。
  3. 第二组用PBS-脂质体处理。
  4. 在实验的不同时间点(例如治疗后2,4,8周),来自每组的一只或两只小鼠将被安乐死以检查炎症和其他有趣的分析。基于我们的实验,FACS分析在治疗2周后,用负载氯膦酸盐的脂质体处理的组中检测到约90%的巨噬细胞减少,并且这种减少保持到实验结束。
  5. 在整个治疗期间,需要密切监测治疗的小鼠的可能的体征和症状,例如体重减轻,黄色,血色或焦油色的粪便,深色尿色,腹泻,疲劳 等。
  6. 在实验结束时(取决于实验),将小鼠安乐死并收集肺组织用于各种分析。

食谱

注意:

  1. 根据公司的指示。
  2. 装载的clodronate 脂质体和含有PBS的脂质体即可使用,但是 通常保持在4℃的溶液需要加热至室温 注射前的温度。
  1. 脂质体
    脂质体是由以下物质制成的人工脂质球:
    1. 磷脂酰胆碱
    2. 胆固醇
  2. 磷酸盐缓冲盐水(PBS)
    1. 10mM Na 2 HPO 4
    2. 10mM NaH 2 PO 4>/
    3. 140mM NaCl

致谢

我们感谢Frederick国家癌症研究实验室的Yinling Hu,Robert H. Wiltrout和Jami Willette-Brown对实验设计的支持和建议。 这项工作由国家癌症研究所(ZIA BC 011212和ZIA BC 011391至Y.H.)支持。

参考文献

  1. Xiao,Z.,Jiang,Q.,Willette-Brown,J.,Xi,S.,Zhu,F.,Burkett,S.,Back,T.,Song,NY,Datla,M.,Sun, ,Goldszmid,R.,Lin,F.,Cohoon,T.,Pike,K.,Wu,X.,Schrump,DS,Wong,KK,Young,HA,Trinchieri,G.,Wiltrout,RH和Hu,Y 。(2013)。 IKKalpha在自发肺鳞状细胞癌发展中的关键作用 Cancer Cell 23(4):527-540。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xiao, Z. and Jiang, Q. (2014). Protocol for Macrophage Depletion from Mice . Bio-protocol 4(11): e1144. DOI: 10.21769/BioProtoc.1144.
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