对疟原虫感染的红细胞进行示踪染色以观察免疫吞噬效应

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

doi:10.21769/BioProtoc.1884

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Peer-reviewed

Cell Tracer Violet and CellTracker Red CMTPX Staining of Purified Mature Plasmodium-infected Red Blood Cells

对疟原虫感染的红细胞进行示踪染色以观察免疫吞噬效应

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.1884 浏览次数: 10791

评审: Pinchas TsukermanAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Feb 2015

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Abstract

Efficient staining methods to identify Plasmodium-infected red blood cells (iRBCs) are crucial to discriminate precisely the immune cells responsible for their elimination from circulation. Here, we describe the protocol for the purification of iRBCs and their subsequent staining with the vital dyes Cell Tracer Violet (CTV) or CellTracker Red CMTPX (CMTPX), both of which readily diffuse into cells and bind covalently to intracellular amines. The iRBCs stained by using this protocol were used in ex vivo phagocytosis assays, to determine the ability of splenic dendritic cells of phagocytizing these parasites (Borges da Silva et al., 2015).

Materials and Reagents

15 ml tubes (TPP, catalog number: 91015 )
1 ml syringes (BD Biosciences, catalog number: 309659 )
B6.Rag2^-/- (RAGKO) mice (Jackson Laboratories, C57BL/6)
Plasmodium chabaudi (AS strain)
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 )
Cell Tracer Violet (CTV) (Thermo Fisher Scientific, catalog number: C34557 )
CellTracker Red CMTPX (CMTPX) (Thermo Fisher Scientific, catalog number: C34552 )
Halothane (Sigma-Aldrich, catalog number: H0150000 )
NaCl
KCl
Na₂HPO₄
KH₂PO₄
Percoll (GE Healthcare Dharmacon, catalog number: 17089101 )
1x phosphate buffered saline (PBS) (see Recipes)
74% Percoll (see Recipes)

Equipment

Centrifuge (Eppendorf, model: 5804 )
1-ml micropipettes
25 gauge needle (BD Biosciences, catalog number: 305122 )
Laminar flow hood (AirClean Systems, catalog number: AC8000HLF )
Epi-Fluorescent microscope (Leica Microsystems, model: DM6000B )

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). Cell Tracer Violet and CellTracker Red CMTPX Staining of Purified Mature Plasmodium-infected Red Blood Cells. Bio-protocol 6(15): e1884. DOI: 10.21769/BioProtoc.1884.
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.