Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay

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 Scientific^TM, 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, Gibco^TM, catalog number: 15140122 )
       
    3. 2-Mercaptoethanol (Thermo Fisher Scientific, Gibco^TM, 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 Plus^TM system Real-Time PCR System (Thermo Fisher Scientific, Applied Biosystems^TM, model: StepOnePlus^TM, catalog number: 4376600 )
   
9. Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: NanoDrop^TM 2000 )
   

Procedure