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

Henrique Borges da Silva; Carlos Eduardo Tadokoro; Maria Regina D’Império Lima

doi:10.21769/BioProtoc.1885

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Peer-reviewed

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

HS Henrique Borges da Silva email

CT Carlos Eduardo Tadokoro

ML Maria Regina D’Império Lima email

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1885 浏览次数: 11027

评审: Pinchas TsukermanAnonymous reviewer(s)

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The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Feb 2015

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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

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

Equipment

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

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Silva, H. B. D., 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.
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.