Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay   

Part I: Quantification of T. cruzi in tissue

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

1. T. cruzi preparation
   1. 5 x 10⁵-1 x 10⁶ LLC-MK₂ cells are seeded into a 15 cm culture dish on day 1 before infection.
      
   2. The LLC-MK₂ cells seeded in step A1 are infected with 2 x 10⁶ T. cruzi strain Tulahuen (MOI = 2).
      
   3. Change the culture medium (20 ml) every 3 days. In Figure 1A, amastigotes are shown in T. cruzi-infected cells (Panel A), which differentiate into the infective form trypomastigotes (Panel B) that are released into the culture medium.
      
   4. After 7 days of infection, trypomastigotes in the culture supernatants are counted microscopically (Figure 1). Approximately 2 x 10⁷ trypomastigotes are obtained.
      
      
      Figure 1. Schematic of T. cruzi in a hemocytometer. A. Amastigotes; B. Trypomastigotes.
      
      
2. Preparation of tissue standards and quantitative PCR
   1. Collect the liver and heart from a non-infected mouse into a 15 cm Petri dish and collect the blood (more than 200 μl) in a CAPIJECT tube.
      
   2. Cut the heart and liver into small pieces by scissor and add 1 x 10⁶ T. cruzi trypomastigotes to 30 mg heart, 30 mg liver, or 200 μl blood from a non-infected C57BL/6 mouse in a 1.5 ml microtube.
      
   3. Add 500 μl lysis buffer (see Recipes) and 5 μl of 10 mg/ml Proteinase K and incubate at 55 °C in a shaking incubator (shaking speed: 110 times/min) for 18 h.
      
   4. Centrifuge at 13,000 x g for 10 min at 4 °C.
      
   5. Transfer the supernatant to a fresh 1.5 ml microtube.
      
   6. Add 500 μl phenol:chloroform:isoamyl alcohol (25:24:1) and mix the contents of the microtube using a vortex.
      
   7. Centrifuge at 13,000 x g for 10 min at 4 °C.
      
   8. Transfer the upper layer to a fresh 1.5 ml microtube.
      
   9. Add 500 μl chloroform and mix the contents of the microtube using a vortex.
      
   10. Centrifuge at 13,000 x g for 10 min at 4 °C.
       
   11. Transfer the upper layer to a fresh 1.5 ml microtube.
       
   12. Add 500 μl 2-propanol and mix the contents of the microtube using a vortex.
       
   13. Centrifuge at 13,000 x g for 10 min at 4 °C.
       
   14. Aspirate the supernatant and wash the pellet in 500 μl 70% ethanol gently.
       
   15. Centrifuge at 5,500 x g for 5 min at 4 °C.
       
   16. Aspirate the supernatant and air dry for 10 min.
       
   17. Dissolve the DNA pellet in TE buffer solution (pH 8.0) and measure the concentration of DNA to generate 25 μg/ml tissue DNA.
       
   18. DNA from the tissue spiked with T. cruzi is sequentially 10-fold diluted with 25 μg/ml DNA from the tissues without T. cruzi (Figure 2). The prepared standards contain DNA from 10³-10^-2 parasites per 50 ng of total DNA. A standard curve to determine the amounts of T. cruzi DNA in the tissues from mice infected with T. cruzi is generated using these standards. The pipet tips should be changed in between each dilution.
       
       
       Figure 2. Scheme of the protocol to generate the standards to quantify T. cruzi in tissue
       
       
   19. For a standard curve to evaluate the amount of TNF-α DNA, 25 mg/ml tissue DNA containing 1 x 10³ T. cruzi (STD1), as prepared above, is subsequently 10-fold diluted with distilled water or TE buffer (Figure 3). Internal control ‘TNF-α DNA’ is used to normalize the amount of tissue being analyzed in each PCR reaction (Cummings and Tarleton, 2003; Caldas et al., 2012). The pipet tips should be changed in between each dilution.
       
       
       Figure 3. Scheme of the protocol to prepare the standards to quantify tissue TNF-α DNA
       
       
   20. Generation of T. cruzi and TNF-α standard calibration curve by qPCR (Figures 4A and 4B)
       1. Mix following components
          10 μl of Go Taq qPCR Master Mix
          1 μl of 10 μM T. cruzi specific primers (Tc1) or TNF-α DNA primers
          2 μl of DNA standards for T. cruzi (Figure 2) or TNF-α DNA (Figure 3)
          7 μl of double distilled water
          
       2. Amplification protocol
          95 °C for 10 min, and 40 cycles of 94 °C for 15 sec and 64.3 °C for 1 min.
          Fluorescent emission (520 nm) is measured at the end of the elongation step. A melting curve phase program is applied with a continuous fluorescent measurement between 65 °C and 95 °C. Each DNA sample is quantified in duplicate from two independent qPCR runs.
          
          
          Figure 4. Standard curve generated with DNA from the livers of non-infected mice spiked with or without T. cruzi trypomastigotes. A and B. A 10-fold dilution series prepared as described above was amplified with primers specific to T. cruzi DNA (A) or murine TNF-α DNA (B). The standard curves were generated from the linear region of each sample application curve.
          
          
3. Quantification of T. cruzi DNA in the tissues from T. cruzi-infected mice
   1. The mice (at least 5 mice) are injected intraperitoneally with 250 μl PBS including 5 x 10² T. cruzi trypomastigotes prepared as in steps A1-A4.
      
   2. At 20 and 30 days after infection, the heart, liver, and blood are collected from mice.
      
   3. 30 mg heart or liver, or 200 μl blood, are incubated with 500 μl lysis buffer/5 μl 10 mg/ml Proteinase K at 55 °C in a shaking incubator (110 min^-1) for 18 h.
      
   4. DNA from the tissues isolated from T. cruzi-infected mice is extracted as described above (steps B4-B17).
      
   5. Normalize the T. cruzi DNA loads in the tissues.
      To normalize the amount of tissue analyzed in each PCR reaction, the murine TNF-α DNA is used to correct for intra-sample variation in the initiation sample amount, sample loading, and DNA recovery (Cummings and Tarleton, 2003). The T. cruzi DNA value and TNF-α DNA value are calculated automatically by plotting the Ct values against each standard of known concentration shown in Figure 4. Normalization of the T. cruzi DNA value = (T. cruzi DNA value/TNF-α DNA value) x 10, where 10 corresponds to the expected value for TNF-α from 30 mg heart/liver or 200 μl blood (Caldas et al., 2012).

Part II: T. cruzi killing assay

Materials and Reagents

1. Pipette tips (Labcon, catalog numbers: 1093-260-000 , 1045-260-000 , 1036-260-000 )
   
2. 10 ml syringe (Terumo, catalog number: SS-10SZP )
   
3. 26 G needle (Terumo, catalog number: NN2613S )
   
4. Cell strainer 40 μm nylon (Corning, Falcon^®, catalog number: 352340 )
   
5. 50 ml tube (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339652 )
   
6. 10 cm culture dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 150466 )
   
7. Scraper (Corning, catalog number: 3008 )
   
8. 24-well plates (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 142475 )
   
9. Glass coverslip (13 mm) (Matsunami Glass, catalog number: C013001 )
   
10. Slide glass (MUTO PURE CHEMICALS, catalog number: 110510 )
    
11. Disposal hemocytometer (All-Biz, catalog number: 4109:37650 )
    
12. 6 cm Petri dishes (AS ONE, catalog number: 1-8549-02 )
    
13. 8-week-old male and female C57BL/6 mice (Japan SLC)
    
14. Trypanosoma cruzi Tulahuen strain (gifted by professor S. Hamano, Institute of Tropical Medicine, Nagasaki University)
    
15. ISOFLURANE Inhalation Solution (Pfizer)
    
16. 70% ethanol (JUNSEI CHEMICAL, catalog number: 17065-0382 )
    
17. HBSS (NACALAI TESQUE, catalog number: 17460-15 )
    
18. 1x phosphate-buffered saline (PBS) (NACALAI TESQUE, catalog number: 14249-24 )
    
19. GM-CSF (Wako Pure Chemical Industries, catalog number: 077-04674 )
    
20. 0.25% trypsin (NACALAI TESQUE, catalog number: 35555-54 ) containing 0.02% EDTA (NACALAI TESQUE, catalog number: 06894-14 )
    
21. IFN-γ (PeproTech, catalog number: 315-05 )
    
22. 4% paraformaldehyde (NACALAI TESQUE, catalog number: 09154-85 )
    
23. Diff-Quik (Sysmex, catalog number: 16920 )
    
24. RPMI 1640 (NACALAI TESQUE, catalog number: 30264-56 )
    
25. ACK lysing buffer (pH 7.2–7.4) (see Recipes)
    1. Ammonium chloride (NH₄Cl) (NAKALAI TESQUE, catalog number: 02423-65 )
       
    2. Potassium hydrogen carbonate (KHCO₃) (Wako Pure Chemical Industries, catalog number: 163-03285 )
       
    3. EDTA (NAKALAI TESQUE, catalog number: 06894-14 )
       
26. RPMI 1640 culture medium (see Recipes)
    1. Fetal bovine serum (Oregon Construction Contractors Board, catalog number: 171012 )
       
    2. Penicillin/streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
       
    3. 2-Mercaptoethanol (Thermo Fisher Scientific, Gibco^TM, catalog number: 21985-023 )
       

Equipment

1. Pipettes (Nichiryo, catalog numbers: 00-NPX2-1000 , 00-NPX2-200 , 00-NPX2-20 , 00-NPX2-2 )
   
2. Scissors (MIZUHO, catalog number: 09-207-00 )
   
3. Forceps (Hammacher, catalog number: HSC_553-11 )
   
4. Microscope (Olympus, model: CX31 )
   
5. Handheld tally counter (Line Seiki, catalog number: H-102 )
   
6. Centrifuge (TOMY SEIKO, model: AX-310 )
   

Procedure

1. Preparation of bone marrow-derived macrophages
   Note: The following protocol referred to a previous report (Weischenfeldt and Porse, 2008).
   1. Euthanize mice with isoflurane inhalation solution.
      
   2. Sterilize the hind legs and abdomen with 70% ethanol.
      
   3. Make an incision in the midline of the abdomen.
      
   4. Clip outward to expose the hind legs.
      
   5. Remove all muscle from the bone using scissors and cut off the bones at the root of the femurs.
      
   6. After cutting the bone at both lower extremities, separate the femur and tibia by cutting at the knee joint in a Petri dish.
      
   7. Flush the bones with HBSS using a 10 ml syringe and a 26 G needle.
      
   8. Pass the bone marrow cells through a cell strainer in 50 ml tubes.
      
   9. Centrifuge at 240 x g for 5 min at 4 °C.
      
   10. Aspirate the supernatant and add 1 ml ACK lysing buffer (see Recipes) for the lysis of red blood cells at room temperature.
       
   11. After 3 min, add 9 ml cold PBS.
       
   12. Centrifuge at 240 x g for 5 min at 4 °C.
       
   13. Aspirate the supernatant.
       
   14. Culture 2 x 10⁷ bone marrow cells isolated from the lower extremities of a mouse in 10 ml RPMI 1640 culture medium (see Recipes) supplemented with 10 ng/ml GM-CSF in a 10 cm culture dish.
       
   15. Wash the cells with PBS twice every 2 days and add fresh RPMI 1640 culture medium with 10 ng/ml GM-CSF.
       
   16. After 6 days, aspirate the supernatant and add 10 ml PBS.
       
   17. Aspirate the PBS and add 1 ml 0.25% trypsin containing 0.02% EDTA. Incubate at 37 °C for 5 min.
       
   18. Add 10 ml RPMI 1640 culture medium and scrape the cells with a scraper.
       
   19. Collect the cells in RPMI 1640 culture medium into 50 ml tubes.
       
   20. Centrifuge at 240 x g for 5 min at 4 °C.
       
   21. Aspirate the supernatant.
       
   22. Place the glass coverslips, sterilized by autoclave treatment, into the wells of a 24-well plate.
       
   23. Plate 5 x 10⁴ bone marrow-derived macrophages on glass coverslips with 500 μl RPMI 1640 culture medium containing 10 ng/ml GM-CSF.
       
   24. After 18 h, bone marrow-derived macrophages are used in the T. cruzi killing assay.
       
       
2. T. cruzi killing assay
   1. Bone marrow-derived macrophages prepared as in steps A23-A25 are cultured with 500 μl RPMI 1640 culture medium for 18 h in the presence of 0, 1, 10, or 100 ng/ml IFN-γ, which enhances parasite-killing activity in phagocytic cells.
      
   2. Aspirate the supernatant and wash with 500 μl PBS five times.
      
   3. Add 500 μl RPMI 1640 culture medium with 10 ng/ml GM-CSF.
      
   4. Infect 5 x 10⁴ T. cruzi trypomastigotes with macrophages for 6 h (MOI = 1).
      
   5. After washing with 500 μl PBS three times, add 500 μl RPMI 1640 culture medium with 10 ng/ml GM-CSF and culture for 72 h.
      
   6. Aspirate the culture medium and wash with 500 μl PBS.
      
   7. Fix cells with 500 μl 4% paraformaldehyde and incubate for 10 min at room temperature.
      
   8. To visualize intracellular amastigotes, stain the cells using Diff-Quik kit.
      
   9. Apply a coverslip onto a slide glass using forceps.
      
   10. Count the number of intracellular amastigotes (Figure 5) under a microscope.
       
       
       Figure 5. The intracellular parasites. Bone marrow-derived macrophages infected with T. cruzi for 6 h in the presence or absence of IFN-γ were rigorously washed and cultured for 72 h. The cells were fixed and stained with Diff-Quik. Original magnification, 400x. * Indicates nucleus. An arrowhead indicates amastigotes.
       
       
   11. The intracellular parasite numbers in 200 macrophages are counted under a light microscope (Figure 6).
       
       
       Figure 6. The number of intracellular amastigotes. The number of parasites per 200 macrophages. Graph represents the mean ± SD from three independent experiments. *P < 0.05. Differences between the control and experimental groups were evaluated using Student’s t-test. Differences of P < 0.05 were considered significant.
       

Recipes

1. Lysis buffer
   100 mM Tris-HCl (pH 8.5)
   5 mM EDTA
   0.2% SDS
   200 mM NaCl
   
2. RPMI 1640 culture medium
   10% fetal bovine serum
   100 μg/ml streptomycin, 100 U/ml penicillin
   100 μM 2-mercaptoethanol
   
3. ACK lysing buffer (pH 7.2-7.4)
   0.15 M NH₄Cl
   1 mM KHCO₃
   0.1 mM EDTA
   

Acknowledgments

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Agency for Medical Research and Development (to K.T. and H. K.), and by the Ichiro Kanehara Foundation and the Kurata Memorial Hitachi Science and Technology Foundation (to H.K.). The protocols provided here were partially modified from previously described methods by S. Cencing et al. (2011) and S. Caldas et al. (2012) (Quantification of T. cruzi) and by J. Weischenfeldt and B. Porse (2008) (Preparation of bone marrow-derived macrophages). We thank Kate Fox, DPhil, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. The authors declare that they have no competing financial interests.

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