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In vivo DCs Depletion with Diphtheria Toxin and MARCO+/MOMA1+ Cells Depletion with Clodronate Liposomes in B6.CD11c-DTR Mice
在B6.CD11c-DTR小鼠中使用白喉毒素和氯膦酸钠脂质体去除树突细胞及MARCO+/MOMA1+细胞

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Abstract

To evaluate precisely the relative roles of different splenic phagocytic cells during an immune response, efficient methods for the depletion of specific populations are needed. Here, we describe the protocols for the depletion of splenic dendritic cells (DCs) by human diphtheria toxin (DTx) treatment in target mice (which express the human DTx receptor in all CD11c+ DCs) and for the specific depletion of MARCO+/MOMA-1+ marginal zone macrophages (MZMΦs) with clodronate liposomes (ClLip) treatment (when a small dose of ClLip is ministered, MZMΦs preferentially uptake ClLip, and clodronate is released inside those cells causing apoptosis-mediated cell death). These protocols are adaptations from previous works (Jung et al., 2002; McGaha et al., 2011), and were used to evaluate the respective roles of DCs and of MZMΦs during the acute phase of experimental blood-stage malaria infection (Borges da Silva et al., 2015).

Materials and Reagents

  1. 25 gauge needle (BD Biosciences, catalog number: 305122 )
  2. Cell strainer (100 μm pore size) (Corning Incorporated, catalog number: 352360 )
  3. 15 ml tubes (TPP, catalog number: 91015 )
  4. 1 ml syringes (BD Biosciences, catalog number: 309659 )
  5. 1 ml syringes with 30 gauge needle (BD Biosciences, catalog number: 328278 )
  6. B6 mice (Jackson Laboratories, model: B6)
  7. B6.CD11c-DTR mice (Jackson Laboratories, model: C57BL/6 )
  8. Diphteria toxin (Sigma-Aldrich, catalog number: D0564 )
  9. Sodium clodronate (Melone Pharmaceutical, catalog number: 22560-50-5 )
  10. RPMI 1640 (Thermo Fisher Scientific, catalog number: 11875093 )
  11. Fetal bovine serum heat inactivated (Thermo Fisher Scientific, catalog number: 10437028 )
  12. Penicilin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140122 )
  13. L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
  14. Sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360070 )
  15. 2-mercaptoethanol Thermo Fisher Scientific, catalog number: 21985023 )
  16. Halothane (Sigma-Aldrich, catalog number: H0150000 )
  17. Monoclonal antibody (mAb) to MARCO (R&D Systems, catalog number: FAB2956P )
  18. mAb to MOMA-1 (Abcam, catalog number: ab51814 )
  19. mAb to F4/80 (eBioscience, catalog number: 47-4801 )
  20. mAb to CD11b (BD Biosciences, catalog number: 562950 )
  21. mAb to CD11c (eBiosciences, catalog number: 17011482 )
  22. mAb to I-Ab (eBiosciences, catalog number: 46532082 )
  23. NaCl
  24. KCl
  25. Na2HPO4
  26. KH2PO4
  27. 1x phosphate buffered saline (PBS) (see Recipes)
  28. Staining Buffer (see Recipes)
  29. Supplemented RPMI 1640 (see Recipes)
  30. ClLip (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5804 )
  2. Laminar flow hood (AirClean Systems, catalog number: AC8000HLF )
  3. FACSCanto II Flow Cytometer, 8-color, lasers blue/red/violet (BD Biosciences, catalog number: 338962 )
  4. Push-Pull syringe pump (KD Scientific, Model: KDS120 )

Procedure

  1. DCs depletion in B6.CD11c-DTR mice
    1. Inject B6.CD11c-DTR mice (6-8 weeks age) i.p. with 2 ng/g of body weight of DTx (200 μl volume per mouse), or with PBS as depletion control (200 μl volume per mouse), using a syringe with 25 gauge needle.
    2. 24 h after injection, mice are euthanized with halothane by inhalation (or other approved euthanasia protocol).
    3. The spleens are removed and processed in a cell strainer inside a sterile culture hood (with 5 ml of supplemented RPMI 1640), followed by two washes with RPMI (at 300 x g, 5 min, 4 °C).
    4. Splenocytes are then stained with mAbs (0.5 µl per 106 cells – each mAb at initial 0.5 mg/ml concentration, diluted in 25 µl of Staining Buffer) to CD11c and I-Ab (1 incubation period of 30 min), re-suspended in staining buffer (200 µl) and analyzed in a FACSCanto device, to evaluate depletion efficiency.

  2. MZMΦs depletion in B6 mice with ClLip
    1. Inject B6 mice (6-8 weeks age) i.v. with 8.5 µg/g of body weight of ClLip (200 µl per mice) prepared as described in (van Rooijen et al., 1993), or with PBS-loaded liposomes (200 µl per mice, 8.5 µg/g of body weight) as depletion control, using a syringe with 30 gauge needle.
    2. 24 h to seven days after injection, mice are euthanized with halothane by inhalation (or other approved euthanasia protocol) and bled by cardiac puncture with a 1 ml syringe with a 25 gauge needle.
      Note: In both time points only MZMΦs are depleted in our protocol (this was done as a control to ensure only MZMΦs are depleted in our protocol, opposite to injection of higher concentrations of ClLip).
    3. The spleens are removed and processed in a cell strainer inside a sterile culture hood (with 5 ml of supplemented RPMI 1640), followed by two washes with RPMI (at 300 x g, 5 min, 4 °C).
    4. Splenocytes are then stained with mAbs (0.5 µl per 106 cells – each mAb at initial 0.5 mg/ml concentration, diluted in 25 µl of Staining Buffer) to MARCO, MOMA-1, CD11b, CD11c and I-Ab (1 incubation period of 30 min), re-suspended in Staining Buffer (200 µl) and analyzed in a FACSCanto device, to evaluate depletion efficiency. An example of results obtained with this experiment is shown in Figure 1A and 1B, respectively.
      Note: Different DC subsets might have different sensitivity to DTx treatment, with different repopulation rates following initial depletion. Thus, for prolonged experiments, an important step would be to perform repopulation rate assays for the DCs subsets of interest.


      Figure 1. In vivo depletion of splenic DCs and MZMΦs. A. Representative plots of mice depleted of DCs using as controls of depletion B6 mice treated with DTX (here, CD11c-DTR mice treated with DTx present with a lower percentage of CD11c+I-A+ DCs, as showed in the gate in the upper right of each histogram (this decrease in percentage is an indicative of cell depletion). Adapted from Borges da Silva et al. (2015). B. Representative plots of mice depleted of MZMΦs using as controls B6 mice treated with ClLip (here, ClLip-treated mice present with a lower percentage of MARCO+MOMA-1+ MZMΦs, as showed in the gates represented in each histogram (this decrease in percentage is an indicative of cell depletion). Adapted from Borges da Silva et al. (2015).

Recipes

  1. 1x phosphate buffered saline (PBS)
    Dissolve the following in 800 ml distilled H2O
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Adjust pH to 7.4
    Adjust volume to 1 L with additional distilled H2O
    Sterilize the solution
    Note: Adjust the pH using HCl and NaOH.
  2. Staining Buffer
    Dissolve the following in 200 ml PBS
    1 ml 10% azide
    2 ml fetal bovine serum heat inactivated
  3. Supplemented RPMI 1640
    Dissolve the following in 200 ml RPMI 1640 medium
    2 ml L-glutamine
    20 ml fetal bovine serum heat inactivated
    2 ml sodium pyruvate
    2 ml Penicilin-Streptomycin
    200 µl 2-mercaptoethanol
  4. ClLip
    1. Inject (0.2 ml/min) an ethereal solution of 50 mg phosphatidylcholine and 8 mg cholesterol into 5 ml of a 50 mM/L clodronate aqueous solution maintained at 42 °C, by using a syringe adapted in a Push-Pull syringe pump, equipped with a fine-gauge needle (No 3D).
    2. During injection, a nitrogen stream will be infused into the clodronate solution, up to liposome formation and removal of residual solvent.
    3. Centrifuge liposome suspension (22,800 x g, 30 min, 25 °C). Wash twice with PBS, and ressuspend in 2 ml PBS.
    4. Filter through a 0.8 Am polycarbonate membrane.

Acknowledgments

HBdS was supported by an award from FAPESP (number: 2014/00810-5) and MRDL was supported by a grant from FAPESP (number: 2013/07140-2), and from CNPq 303676/2014-0 (MRDL) and 448765/2014-4 (MRDL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The DTX-mediated depletion of DCs was adapted from Jung et al. (2002), and the ClLip-mediated depletion of MZMΦs was adapted from McGaha et al. (2011).

References

  1. Borges da Silva, H., Fonseca, R., Cassado Ados, A., Machado de Salles, E., de Menezes, M. N., Langhorne, J., Perez, K. R., Cuccovia, I. M., Ryffel, B., Barreto, V. M., Marinho, C. R., Boscardin, S. B., Alvarez, J. M., D'Imperio-Lima, M. R. and Tadokoro, C. E. (2015). In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria. PLoS Pathog 11(2): e1004598.
  2. Jung, S., Unutmaz, D., Wong, P., Sano, G., De los Santos, K., Sparwasser, T., Wu, S., Vuthoori, S., Ko, K., Zavala, F., Pamer, E. G., Littman, D. R. and Lang, R. A. (2002). In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17(2): 211-220.
  3. McGaha, T. L., Chen, Y., Ravishankar, B., van Rooijen, N. and Karlsson, M. C. (2011). Marginal zone macrophages suppress innate and adaptive immunity to apoptotic cells in the spleen. Blood 117(20): 5403-5412.
  4. van Rooijen, N. and van Kesteren-Hendrikx, E. (2003). "In vivo" depletion of macrophages by liposome-mediated "suicide". Methods Enzymol 373: 3-16.

简介

为了准确地评估不同的脾吞噬细胞在免疫应答期间的相对作用,需要用于消耗特定群体的有效方法。 在这里,我们描述了通过人类白喉毒素(DTx)治疗在目标小鼠(其在所有CD11c + DC中表达人类DTx受体)中脾脏树突状细胞(DCs) 用氯膦酸脂质体(ClLip)处理(当小剂量的ClLip被分配时,MZMΦs优先摄取)的MARCO + /MOMA-1 + 边缘区巨噬细胞(MZMΦ) ClLip和氯膦酸盐在那些细胞内释放,引起凋亡介导的细胞死亡)。 这些方案是来自先前作品的改编(Jung等人,2002; McGaha等人,2011),并且用于评价DC和MZMΦ的各自的作用 在实验性血液阶段疟疾感染的急性期期间(Borges da Silva等人,2015)。

材料和试剂

  1. 25号针(BD Biosciences,目录号:305122)
  2. 细胞过滤器(100μm孔径)(Corning Incorporated,目录号:352360)
  3. 15ml管(TPP,目录号:91015)
  4. 1ml注射器(BD Biosciences,目录号:309659)
  5. 1ml具有30号针头的注射器(BD Biosciences,目录号:328278)
  6. B6小鼠(Jackson Laboratories,型号:B6)
  7. B6.CD11c-DTR小鼠(Jackson Laboratories,型号:C57BL/6)
  8. 白喉毒素(Sigma-Aldrich,目录号:D0564)
  9. 氯化钠(Melone Pharmaceutical,目录号:22560-50-5)
  10. RPMI 1640(Thermo Fisher Scientific,目录号:11875093)
  11. 胎牛血清热灭活(Thermo Fisher Scientific,目录号:10437028)
  12. 青霉素 - 链霉素(Thermo Fisher Scientific,目录号:15140122)
  13. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030081)
  14. 丙酮酸钠(Thermo Fisher Scientific,目录号:11360070)
  15. 2-巯基乙醇Thermo Fisher Scientific,目录号:21985023)
  16. 氟烷(Sigma-Aldrich,目录号:H0150000)
  17. MARCO的单克隆抗体(mAb)(R& D Systems,目录号:FAB2956P)
  18. mAb至MOMA-1(Abcam,目录号:ab51814)
  19. mAb至F4/80(eBioscience,目录号:47-4801)
  20. mAb到CD11b(BD Biosciences,目录号:562950)
  21. mAb到CD11c(eBiosciences,目录号:17011482)
  22. mAb至I-Ab(eBiosciences,目录号:46532082)
  23. NaCl
  24. KCl
  25. Na HPO 4
  26. KH 2 PO 4
  27. 1×磷酸盐缓冲盐水(PBS)(参见Recipes)
  28. 染色缓冲液(参见配方)
  29. 补充的RPMI 1640(见配方)
  30. ClLip(参见食谱)

设备

  1. 离心机(Eppendorf,型号:5804)
  2. 层流罩(AirClean Systems,目录号:AC8000HLF)
  3. FACSCanto II流式细胞仪,8色,激光蓝/红/紫(BD Biosciences,目录号:338962)
  4. 推拉注射泵(KD Scientific,型号:KDS120)

程序

  1. 在B6.CD11c-DTR小鼠中DCs耗尽
    1. 注射B6.CD11c-DTR小鼠(6-8周龄)i.p 。用2ng/g体重的DTx(每只小鼠200μl体积),或用PBS作为耗尽对照(每只小鼠200μl体积),使用具有25号针头的注射器。
    2. 注射后24小时,通过吸入(或其他批准的安乐死方案)用氟烷安乐死小鼠。
    3. 取出脾并在无菌培养罩(用5ml补充的RPMI 1640)内的细胞过滤器中处理,然后用RPMI洗涤两次(300×g,5分钟,4℃) 。
    4. 然后将脾细胞用mAbs(0.5μl/10 6个细胞 - 每个mAb,初始0.5mg/ml浓度,在25μl的染色缓冲液中稀释)稀释到CD11c和I-Ab(1个孵育期30分钟),重悬于染色缓冲液(200μl)中并在FACSCanto装置中分析,以评价消耗效率。
  2. 使用ClLip的B6小鼠中的MZMΦs耗尽
    1. 注射B6小鼠(6-8周龄)i.v 。 (van Rooijen等人,1993)中所述制备的8.5μg/g体重的ClLip(每只小鼠200μl),或用PBS装载的脂质体(每只小鼠200μl,8.5μg/μg/g体重)作为消耗对照,使用具有30号针头的注射器
    2. 注射后24小时至7天,通过吸入(或其他批准的安乐死方案)用氟烷安乐死小鼠,并用具有25号针的1ml注射器通过心脏穿刺放血。
      注意:在两个时间点,在我们的方案中仅消耗MZMΦ(这是作为对照进行的,以确保在我们的方案中仅消耗MZMΦ,与注射更高浓度的ClLip相反)。
    3. 取出脾并在无菌培养罩(用5ml补充的RPMI 1640)内的细胞过滤器中处理,然后用RPMI洗涤两次(300×g,5分钟,4℃) 。
    4. 然后将脾细胞用mAb(0.5μl/10 6个细胞 - 每个mAb,初始浓度为0.5mg/ml,稀释在25μl的染色缓冲液中)染色至MARCO,MOMA-1,CD11b,CD11c和I-Ab(1个孵育时间为30分钟),重悬于染色缓冲液(200μl)中并在FACSCanto装置中分析,以评估消耗效率。用该实验获得的结果的实例分别示于图1A和1B中 注意:不同的DC子集可能对DTx治疗具有不同的敏感性,在初始消耗后具有不同的再生速率。因此,对于长时间的实验,一个重要的步骤将是对感兴趣的DCs亚群进行繁殖率测定。


      在脾脏DC和MZMΦ的体内耗尽 A.使用DTX处理的??枯竭B6小鼠作为对照的DC消耗的小鼠的代表性图这里,如在每个直方图的右上方的门中所示,用DTx处理的CD11c-DTR小鼠存在具有较低百分比的CD11c + IA + DC使用ClLip处理的B6小鼠(在此为ClLip),作为对照的用MZMΦ消耗的小鼠的代表性图处理的小鼠存在具有较低百分比的MARCO + MOMA-1 + MZMΦs,如在每个直方图中表示的门中所示(这个百分比的减少是细胞的指示耗尽),改编自博尔赫斯·达席尔瓦等人(2015)。

食谱

  1. 1×磷酸盐缓冲盐水(PBS)
    将以下物质溶于800ml蒸馏H 2 O中 8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4 sub/
    用额外的蒸馏H 2 O 2调节体积至1L 消毒溶液
    注意:使用HCl和NaOH调整pH值。
  2. 染色缓冲液
    将以下物质溶于200ml PBS中
    1ml 10%叠氮化物 2ml胎牛血清热灭活
  3. 补充RPMI 1640
    将下列物质溶于200ml RPMI 1640培养基中 2ml L-谷氨酰胺 20ml胎牛血清热灭活
    2 ml丙酮酸钠 2ml青霉素 - 链霉素 200μl2-巯基乙醇
  4. ClLip
    1. 通过使用适合于推拉式注射泵的注射器,将50mg磷脂酰胆碱和8mg胆固醇的醚溶液注射(0.2ml/min)到保持在42℃的5ml 50mM/L氯膦酸盐水溶液中使用细针(N o
    2. 在注射期间,氮气流将注入氯膦酸盐溶液中,直到形成脂质体并除去残余溶剂。
    3. 离心脂质体悬浮液(22,800×g/30分钟,25℃)。用PBS洗涤两次,并悬浮在2ml PBS中
    4. 通过0.8μm聚碳酸酯膜过滤。

致谢

HBDS获得FAPESP(编号:2014/00810-5)的奖励支持,MRDL由FAPESP(编号:2013/07140-2)和CNPq 303676/2014-0(MRDL)和448765/2014-4(MRDL)。资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。 DTX介导的DC的耗竭源自Jung等人。 (2002),并且ClLip介导的MZMΦ的消耗从McGaha等人改编。 (2011)。

参考文献

  1. Borges da Silva,H.,Fonseca,R.,Cassado Ados,A.,Machado de Salles,E.,de Menezes,MN,Langhorne,J.,Perez,KR,Cuccovia,IM,Ryffel,B.,Barreto, VM,Marinho,CR,Boscardin,SB,Alvarez,JM,D'Imperio-Lima,MR和Tadokoro,CE(2015)。  体内方法揭示了DC在CD4 + T细胞活化和寄生虫清除期间的关键作用实验性血液阶段疟疾的急性期。 PLoS Pathog 11(2):e1004598。
  2. Jung,S.,Unutmaz,D.,Wong,P.,Sano,G.,De los Santos,K.,Sparwasser,T.,Wu,S.,Vuthoori,S.,Ko,K.,Zavala,F 。,Pamer,EG,Littman,DRand Lang,RA(2002)。  CD11c + 树突状细胞的体内消耗消除了外源性细胞相关抗原对CD8 + sup/T细胞的引发。 a> 免疫力 17(2):211-220
  3. McGaha,TL,Chen,Y.,Ravishankar,B.,van Rooijen,N.和Karlsson,MC(2011)。  "in vivo"通过脂质体介导的"自杀"消耗巨噬细胞。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Silva, H. B., Tadokoro, C. E. and D’Império-Lima, M. R. (2016). In vivo DCs Depletion with Diphtheria Toxin and MARCO+/MOMA1+ Cells Depletion with Clodronate Liposomes in B6.CD11c-DTR Mice. Bio-protocol 6(15): e1885. DOI: 10.21769/BioProtoc.1885.
  2. Borges da Silva, H., Fonseca, R., Cassado Ados, A., Machado de Salles, E., de Menezes, M. N., Langhorne, J., Perez, K. R., Cuccovia, I. M., Ryffel, B., Barreto, V. M., Marinho, C. R., Boscardin, S. B., Alvarez, J. M., D'Imperio-Lima, M. R. and Tadokoro, C. E. (2015). In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria. PLoS Pathog 11(2): e1004598.
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