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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

发布: 2017年11月20日第7卷第22期 DOI: 10.21769/BioProtoc.2613 浏览次数: 8098

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) search Quantitative PCR (定量PCR) search Killing assay (杀灭试验) search Mouse (小鼠) search Heart (心脏) search Liver (肝脏) search Blood (血液) search Bone marrow-derived macrophage (源自骨髓的巨噬细胞) search

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

  1. Pipette tips (Labcon, catalog numbers: 1093-260-000 , 1045-260-000 , 1036-260-000 )
  2. 15 cm culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150468 )
  3. Blood collection tubes CAPIJECT (Terumo, catalog number: CJ-NA )
  4. 10 cm Petri dishes (Sansyo, catalog number: 36-3406 )
  5. 1.5 ml microtubes (FUKAEKASEI and WATSON, catalog number: 131-415C )
  6. Disposal hemocytometer (All-Biz, catalog number: 4109:37650 )
  7. 8-week-old male and female C57BL/6 mice (Japan SLC)
  8. 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
  9. Trypanosoma cruzi Tulahuen strain (gifted by professor S. Hamano (Institute of Tropical medicine, Nagasaki University))
  10. 10 mg/ml Proteinase K (Merck, catalog number: 124568 )
  11. Phenol:chloroform:isoamyl alcohol (25:24:1) (NACALAI TESQUE, catalog number: 25970-56 )
  12. Chloroform (JUNSEI CHEMICAL, catalog number: 28560-0330 )
  13. 2-Propanol (JUNSEI CHEMICAL, catalog number: 64605-0330 )
  14. 70% ethanol (JUNSEI CHEMICAL, catalog number: 17065-0382 )
  15. TE buffer solution (pH 8.0) (NACALAI TESQUE, catalog number: 32739-31 )
  16. Go Taq qPCR Master Mix (Promega, catalog number: A6002 )
  17. RPMI 1640 (NACALAI TESQUE, catalog number: 30264-56 )
  18. 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)
  19. 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 )
  20. RPMI 1640 culture medium (see Recipes)
    1. Fetal bovine serum (CCB, catalog number: 171012 )
    2. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    3. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985023)

Equipment

  1. Pipettes (Nichiryo, catalog numbers: 00-NPX2-1000 , 00-NPX2-200 , 00-NPX2-20 , 00-NPX2-2 )
  2. Forceps (Hammacher, catalog number: HSC_553-11 )
  3. Microbalance (Chyo Balance, model: JPN-200W )
  4. Scissor (Fine Science Tools, catalog number: 91460-11 )
  5. Shaking incubator (TAITEC, model: BR-43FM MR )
  6. Centrifuge (TOMY SEIKO, model: MX-200 )
  7. Vortex
  8. Real-Time PCR thermal cycler: Step One PlusTM system Real-Time PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOnePlusTM, catalog number: 4376600 )
  9. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

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分类

微生物学 > 抗微生物试验 > 杀伤试验

免疫学 > 动物模型 > 小鼠

细胞生物学 > 组织分析 > 组织分离

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