组织中克氏锥虫的定量及克氏锥虫杀灭试验

Hisako Kayama; Shoko Kitada; Kiyoshi Takeda

doi:10.21769/BioProtoc.2613

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Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay

组织中克氏锥虫的定量及克氏锥虫杀灭试验

HK Hisako Kayama

SK Shoko Kitada

KT Kiyoshi Takeda email

发布: 2017年11月20日第7卷第22期 DOI: 10.21769/BioProtoc.2613 浏览次数: 7432

评审: Alexandros AlexandratosAnonymous reviewer(s)

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Abstract

Infection with Trypanosoma cruzi causes Chagas disease. The methods provided here allow for the quantification of T. cruzi in the liver, heart, and blood of intraperitoneally-infected mice and analysis of the killing activity of the cells infected with T. cruzi in vitro.

Keywords: Trypanosoma cruzi (克氏锥虫)

Quantitative PCR (定量PCR)

Bone marrow-derived macrophage (源自骨髓的巨噬细胞)

Background

Chagas disease, characterized by chronic cardiomyopathy, is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi (Bonney et al., 2015). Approximately 20 million people in Latin America suffer from Chagas disease (Ribeiro et al., 2012; Flavia Nardy et al., 2015), and it has become a global health issue owing to the migration of infected individuals (Andrade et al., 2014; Garcia et al., 2015; Requena-Mendez et al., 2015). Several drugs, such as nifurtimox and benznidazole, have been developed for treating Chagas disease. However, these drugs need to be taken for several months and severe side effects have been reported (Viotti et al., 2009). A major aim of treatment is to inhibit T. cruzi transmission via blood as well as prevent the development of heart failure. Thus, the protocol for quantification of T. cruzi and the T. cruzi killing assay presented here might aid the development of novel diagnostic methods and therapeutic strategies for Chagas disease.

Part I: Quantification of T. cruzi in tissue

The following protocol (Kitada et al., 2017) was partially modified from the previously described methods (Cencig et al., 2011; Caldas et al., 2012).

Materials and Reagents

Pipette tips (Labcon, catalog numbers: 1093-260-000 , 1045-260-000 , 1036-260-000 )
15 cm culture dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 150468 )
Blood collection tubes CAPIJECT (Terumo, catalog number: CJ-NA )
10 cm Petri dishes (Sansyo, catalog number: 36-3406 )
1.5 ml microtubes (FUKAEKASEI and WATSON, catalog number: 131-415C )
Disposal hemocytometer (All-Biz, catalog number: 4109:37650 )
8-week-old male and female C57BL/6 mice (Japan SLC)
LLC-MK₂ cells (Rhesus monkey kidney epithelial cells) gifted by Professor S. Hamano (Institute of Tropical Medicine, Nagasaki University), which are also available at ATCC. Information regarding the LLC-MK₂ cells is available at https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Kidney/CCL-7.aspx#documentation
Trypanosoma cruzi Tulahuen strain (gifted by professor S. Hamano (Institute of Tropical medicine, Nagasaki University))
10 mg/ml Proteinase K (Merck, catalog number: 124568 )
Phenol:chloroform:isoamyl alcohol (25:24:1) (NACALAI TESQUE, catalog number: 25970-56 )
Chloroform (JUNSEI CHEMICAL, catalog number: 28560-0330 )
2-Propanol (JUNSEI CHEMICAL, catalog number: 64605-0330 )
70% ethanol (JUNSEI CHEMICAL, catalog number: 17065-0382 )
TE buffer solution (pH 8.0) (NACALAI TESQUE, catalog number: 32739-31 )
Go Taq qPCR Master Mix (Promega, catalog number: A6002 )
RPMI 1640 (NACALAI TESQUE, catalog number: 30264-56 )
Primers (Invitrogen custom DNA primers)
T. cruzi specific primers (Tc1), 5’-cgagctgttgcccacacgggtgct-3’ and 5’-cctccaagcagcggatagttcagg-3’ (Cencig et al., 2011); and TNF-α DNA primers, 5’-tccctctcatcagttctatggccca-3’ and 5’-cagcaagcatctatgcacttagacccc-3’ (Caldas et al., 2012)
Lysis buffer (see Recipes)
1. Tris-HCl (NACALAI TESQUE, catalog number: 35434-21 )
2. Ethylenediaminetetraacetate acid (EDTA) (NACALAI TESQUE, catalog number: 06894-14 )
3. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 311-90271 )
4. Potassium chloride (NaCl) (JUNSEI CHEMICAL, catalog number: 19015-0350 )
RPMI 1640 culture medium (see Recipes)
1. Fetal bovine serum (CCB, catalog number: 171012 )
2. Penicillin/streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
3. 2-Mercaptoethanol (Thermo Fisher Scientific, Gibco^TM, catalog number: 21985023)

Equipment

Pipettes (Nichiryo, catalog numbers: 00-NPX2-1000 , 00-NPX2-200 , 00-NPX2-20 , 00-NPX2-2 )
Forceps (Hammacher, catalog number: HSC_553-11 )
Microbalance (Chyo Balance, model: JPN-200W )
Scissor (Fine Science Tools, catalog number: 91460-11 )
Shaking incubator (TAITEC, model: BR-43FM MR )
Centrifuge (TOMY SEIKO, model: MX-200 )
Vortex
Real-Time PCR thermal cycler: Step One Plus^TM system Real-Time PCR System (Thermo Fisher Scientific, Applied Biosystems^TM, model: StepOnePlus^TM, catalog number: 4376600 )
Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: NanoDrop^TM 2000 )

Procedure

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Kayama, H., Kitada, S. and Takeda, K. (2017). Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay. Bio-protocol 7(22): e2613. DOI: 10.21769/BioProtoc.2613.