Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi
克氏锥虫中利用胸苷类似物进行DNA交换分析   

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Scientific Reports
Sep 2018

 

Abstract

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5′-chloro-2′-deoxyuridine (CldU) and 5′-iodo-2′-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

Keywords: DNA exchange (DNA交换), Genetic exchange (遗传交换), Thymidine analogs (胸苷类似物), CldU (CldU), IdU (IdU), DNA replication (DNA复制), Trypanosomatids (锥虫属), Trypanosoma cruzi (克氏锥虫)

Background

Trypanosomatids parasites are single-celled eukaryotes within the supergroup Excavata (Adl et al., 2012). Among them, there are human pathogens responsible for causing several devastating diseases such as T. cruzi (etiological agent of Chagas disease, also called American trypanosomiasis), T. brucei (etiological agent of sleeping sickness, also called Human African trypanosomiasis), and Leishmania spp. (etiological agent of distinct forms of leishmaniasis). Altogether, these peculiar organisms are responsible for more than 50,000 deaths annually worldwide (Ponte-Sucre, 2016; Browne et al., 2017; da Silva et al., 2017b; Torres-Guerrero et al., 2017). Although trypanosomatids predominantly multiply by clonal generation through longitudinal binary fission, the presence of DNA exchange in these organisms, including T. cruzi and Leishmania spp., have been debated over the years (Gaunt et al., 2003; Alves et al., 2018). Due to the lack of practical and straightforward experiments and due to the difficulty of reproducing complex assays, DNA exchange still little explored, even though it may represent a novel and unknown form that trypanosomatids use to gain variability. However, a recent work developed by our group presented an elegant approach to detect DNA exchange in different strains of T. cruzi (Alves et al., 2018). In this study, we figured out higher rates of DNA exchange in naturally-occurring hybrid CL Brener strain (TcIV) relative to naturally-occurring non-hybrid Y strain (TcII). Also, this work pointed out that the recombinase Rad51 contributes significantly to the efficiency of this mechanism (Alves et al., 2018).

Here, we describe in detail the protocol used in this study to detect DNA exchange in trypanosomatids. We named it as Analysis of DNA Exchange using Thymidine Analogs (ADExTA). The principle of the method is based on the incorporation of two distinguishable halogenated thymidine analogs [e.g., 5′-chloro-2′-deoxyuridine (CldU) and 5′-iodo-2′-deoxyuridine (IdU)] into DNA during the S phase of the cell cycle. The presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies after a DNA denaturation step using HCl, which will generate distinct signals after a secondary reaction using fluorochrome-labeled antibodies. This principle is the same used in the technique single-molecule analysis of DNA replication (Herrick and Bensimon, 1999), usually called DNA combing (Calderano et al., 2015; Stanojcic et al., 2016; da Silva et al., 2018). The method is rapid, relatively simple and provides a good overview of the presence of DNA exchange, as well as its rate. Of note, this protocol represents a considerable advantage when compared to other laborious approaches that require harsh genetic manipulation and is time-consuming, such as constructions using recombinant DNA to introduce distinct drug-resistance markers in different cells. By using this method, the presence of DNA exchange and its measurement can be carried out using a standard fluorescence microscope. Theoretically, this protocol can be applied to any organism that allows the incorporation of CldU/IdU, and it typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, although thymidine analogs can be toxic for some cell types (Davidson and Kaufman, 1979; Hancock et al., 2009), in trypanosomatids they have been used apparently without showing any toxic effect (Elias et al., 2007; da Silva et al., 2013; Stanojcic et al., 2016; da Silva et al., 2017a).

Materials and Reagents

  1. Microtubes 1.5 ml (Axygen, Maxyclear, catalog number: MCT-150-C-S)
  2. Centrifuge tubes 15 ml (Corning, catalog number: CLS430791)
  3. Centrifuge tubes 50 ml (Corning, catalog number: CLS430290)
  4. Culture flasks 25 cm2 (Corning, canted neck, cap plug seal, catalog number: CLS430168)
  5. Syringe filter 0.22 μm (Sartori, Minisart Syringe filter, catalog number: 16534)
  6. Micropipette tips, 10 μl, 200 μl and 1,000 μl (Axygen, catalog numbers: T-300, T-200-Y and T-1000-B)
  7. Serological pipettes, 10 ml (Costar Sterile, catalog number: 4488)
  8. Microscope Slides (Knittel glass, non-color, catalog number: VA111101FKB.01)
  9. Coverslips (Knitell glass, 22 x 22 mm, catalog number: VD12222Y1A.01)
  10. Plastic coverslips (use a cut plastic pocket for binder)
  11. T. cruzi cell lines: Y strain (TcII) and CL Brener strain (TcIV)
  12. 5′-Iodo-2′-deoxyuridine (IdU) (Sigma-Aldrich, catalog number: I7125)
  13. 5′-Chloro-2′-deoxyuridine (CldU) (Abcam, catalog number: ab213715)
  14. Mouse α-IdU monoclonal antibody (BD, catalog number: 347580)
  15. Rat α-CldU monoclonal antibody (Accurate, catalog number: YSRTMCA2060GA)
  16. Goat α-mouse IgG1 secondary antibody, Alexa Fluor 568 (Thermo Scientific, catalog number: A-21124)
  17. Goat α-rat IgG (H + L) secondary antibody, Alexa Fluor 488 (Thermo Scientific, catalog number: A-11006)
  18. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127)
  19. Poly-L-lysine hydrochloride (Sigma-Aldrich, catalog number: P2658)
  20. Bovine serum albumin (Sigma-Aldrich, catalog number: 05470)
  21. Triton X-100 (Sigma-Aldrich, catalog number: T8787)
  22. NaCl (Sigma-Aldrich, catalog number: S9888)
  23. KCl (Sigma-Aldrich, catalog number: P9541)
  24. Na2HPO4 (Sigma-Aldrich, catalog number: 255793)
  25. KH2PO4 (Merck, catalog number: 104873)
  26. D-Glucose (Sigma-Aldrich, catalog number: G8270)
  27. Liver Infusion broth (BD, catalog number: 226920)
  28. Tryptose (Sigma-Aldrich, catalog number: 70937)
  29. Hemin (Sigma-Aldrich, catalog number: H9039)
  30. Triethanolamine (Sigma-Aldrich, catalog number: 90279)
  31. Fetal Bovine Serum (Fisher Scientific, catalog number: 10500056)
  32. Penicillin G sodium salt (Sigma-Aldrich, catalog number: 13752)
  33. Streptomycin sulfate salt (Sigma-Aldrich, catalog number: S9137)
  34. Boric Acid (Sigma-Aldrich, catalog number: B6768)
  35. Sodium tetraborate (Sigma-Aldrich, catalog number: 221732)
  36. NaOH (Merck, catalog number: 1064980500)
  37. Vectashield Mounting Medium with DAPI (Vector Labs, catalog number: H-1200)
  38. Hydrochloric acid fuming 37% (Merck, catalog number: 100317)
  39. Nail varnish (any brand, preferably colorless)
  40. Alkalinized water (see Recipes)
  41. Phosphate buffered saline (1x PBS) (see Recipes)
  42. Liver Infusion Tryptose (LIT) medium (see Recipes)
  43. 5′-chloro-2′-deoxyuridine solution (CldU-S) (see Recipes)
  44. 5′-iodo-2′-deoxyuridine solution (IdU-S) (see Recipes)
  45. Fixation buffer (FB) (see Recipes)
  46. Poly-L-lysine solution (PLS) (see Recipes)
  47. Permeabilization solution (PS) (see Recipes)
  48. Denaturation buffer (DB) (see Recipes)
  49. Neutralization buffer (NB) (see Recipes)
  50. Blocking solution (BS) (see Recipes)

Equipment

  1. Microcentrifuge (Eppendorf, model: 5424 R)
  2. Motorized pipet dispenser (Fisher Scientific, Fisherbrand, catalog number: 03-692-172)
  3. Water bath (Cientec, model: CT-226)
  4. Incubator BOD (Vitrex, model: NI1705)
  5. Fluorescence Microscope [Olympus, model: BX51, coupled to an XM10 digital camera. Filters specifications: U-MWU2 (excitation = 330-385 nm; emission = 420 nm), U-MWIBA3 (excitation = 460-495 nm; emission = 510-550 nm), and U-MWG2 (excitation = 510-550 nm; emission = 590 nm)]
  6. Micropipettes (Gilson, models: Pipetman P10, P20, P200 and P1000)
  7. Centrifuge (Eppendorf, model: 5810 R), equipped with 4 x 250 ml Swing-Bucket Rotor
  8. Neubauer chamber with cover glass (Sigma-Aldrich, model: Bright-LineTM Hemacytometer)
  9. Biosafety Class II A2 cabinet (Pachane, model: PA 700)
  10. pH meter (Gehaka, model: PG1800)
  11. Autoclave

Software

  1. Olympus Cell F software (Olympus, version 5.1.2640)
  2. ImageJ (NIH, version 1.47t)
  3. Microsoft Excel (Microsoft Office-any version) or GraphPad Prism (GraphPad software, Inc.)

Procedure

- Before starting with this protocol, we recommend reading it completely, especially the Notes section at the end of this article.
- Cells should be cultured in medium and growth conditions that are appropriate for the given cell type. To achieve a suitable number of labeled cells during data analysis, they must be in the exponential phase.
- Although the thymidine analogs can be toxic when used in high dosages for long periods of incorporation, we did not detect any alterations regarding toxicity in our T. cruzi cell lines during our analysis.
- Important: If the antibodies that will be used in this protocol have not been previously tested for specificity, we strongly recommend that Procedure B (controls for checking the specificity of the antibodies) be performed first.


  1. Analysis of the DNA exchange
    1. Incubate T. cruzi cells (28 °C) until they reach exponential phase (~1 x 107 cells/ml) in 10 ml of culture. The parasites density varies according to the lineage used.
      Note: In our assays, we used two different lineages: T. cruzi Y strain (TcII), and T. cruzi CL Brener strain (TcIV), both in epimastigote forms. The exponential phase may vary according to each strain.
    2. Split the exponential T. cruzi culture into two culture flasks (25 cm2) adding 5 ml per flask.
    3. Thaw both solutions of thymidine analogs CldU-S and IdU-S at room temperature.
    4. Add one of the thymidine analogs (e.g., CldU) to a final concentration of 100 μM in one flask, and the other one (e.g., IdU) in the remaining flask, also to a final concentration of 100 μM. 
    5. Incubate each flask containing the different thymidine analogs for 12 h at 28 °C.
      Note: The incubation time, as well as the temperature, may vary according to each parasite strain or cell type. In our protocol, the incubation time (period of thymidine analogs incorporation) was determined empirically after a long period of testing. We managed to get good results using a period corresponding to the half of the doubling time of the lineage used (T. cruzi doubling time is 24 h). 
    6. Warm an aliquot of 200 ml of Liver Infusion Tryptose (LIT) medium at 28 °C.
      Note: If it is available, use a water bath.
    7. Collect the 5 ml of T. cruzi parasites CldU- or IdU-incorporated by centrifugation at 800 x g for 5 min (from Step A6). 
    8. Remove the medium from cells carefully and replace it with 10 ml of LIT at 28 °C.
    9. Suspend the pellet carefully and repeat the Steps A7-A8 twice for a total of three washes with LIT. For the last wash, suspend the pellet carefully into 5 ml LIT.
    10. Mix the both CldU- and IdU-incorporated T. cruzi cultures into one single flask, totalizing a volume of 10 ml.
    11. Incubate for 24 h at 28 °C.
      Note: The incubation time, as well as the temperature, may vary according to each parasite strain or cell type.
    12. Chill the 1x PBS to 4 °C.
    13. Harvest the cells by centrifugation at 800 x g for 5 min at 4 °C and wash twice each using 5 ml of cold 1x PBS.
    14. Remove the 1x PBS from cells carefully to preserve the pellet.
    15. Suspend the pellet in 1 ml of cold Fixation buffer (FB) and transfer to a 1.5 ml microcentrifuge tube.
    16. Incubate at 4 °C for 7 min and wash three times using 5 ml of cold 1x PBS (centrifuging at 800 x g for 5 min in each wash) each. For the last wash, suspend the pellet carefully into 500 μl of 1x PBS. If the pellet is too small (i.e., almost invisible to the naked eye), decrease the volume of the last wash to 100 μl of 1x PBS.
    17. Prepare the slides to receive the T. cruzi cells by spreading 2.5 μl of Poly-L-lysine solution (PLS) onto slide surface until PLS dry out. Prepare three slides for each sample from Step A15.
      Note: Use a coverslip to help spreading the PLS onto slide (see Figure 1 for more detail).


      Figure 1. Illustration to clarify the Step A17. A coverslip can be used to help to spread out the PLS drop (2.5 μl) onto slides.

    18. Spread the suspended-pellet (from Step A16) carefully in each of the three slides. Use 25-30 μl and save the remaining suspended-pellet volume in case you need to remake some slides.
      Notes: 
      1. Use the same surface where PLS was previous spread.
      2. Each slide is one technical replicate.
    19. Wait for the cells to precipitate and settle on slide for 10-15 min at room temperature. Ensure that the cells do not dry out.
    20. Permeabilize the cells by adding 50 μl of Permeabilization solution (PS) for 10 min at room temperature.
    21. Wash the slide containing cells three times using 1x PBS.
      Note: Use a P1000 micropipette to spread (by sneezing) 1x PBS (1 ml) onto slide three times.
    22. Denature the DNA of the cells by adding 50 μl of Denaturation buffer (DB) for 20 min at room temperature.
      Note: Use a plastic coverslip to help DB spread out and avoid dry out.
    23. Remove the plastic coverslip and wash the slide containing cells once using 1x PBS. 
    24. Neutralize the reaction by adding 50 μl of Neutralization buffer (NB) for 10 min at room temperature.
      Note: Use a plastic coverslip to help NB spread out.
    25. Remove the plastic coverslip and wash the slide containing cells three times using 1x PBS. 
    26. Dilute both primary antibodies [i.e., rat α-CldU monoclonal antibody (Accurate) and mouse α-IdU monoclonal antibody (BD)] 1:300 in Blocking solution (BS).
    27. Spread out 50 μl of the diluted primary antibodies on the slide-surface containing cells (from Step A25). Incubate for 2 h at room temperature.
      Note: Use a plastic coverslip to help primary antibodies spread out and avoid dry out. 
    28. Remove the plastic coverslip and wash the slide containing cells three times using 1x PBS.
    29. Dilute both secondary antibodies [i.e., Alexa Fluor 568-conjugated goat α-mouse (Thermo Scientific) and Alexa Fluor 488-conjugated goat α-rat (Thermo Scientific)] 1:300 in BS.
      Note: From this step onwards, do not expose the slides to light.
    30. Spread out 50 μl of the diluted secondary antibodies on the slide-surface containing cells (from Step A28). Incubate for 2 h at room temperature.
      Note: Use a plastic coverslip to help primary antibodies spread out and avoid dry out.
    31. Remove the plastic coverslip and wash the slide containing cells three times using 1x PBS.
    32. Ensure remove of all the liquid from the slide surface containing cells and add 4 μl of Vectashield mounting medium containing DAPI.
      Note: This solution is used as the anti-fade mounting solution and to stain organelles containing DNA.
    33. Add a glass coverslip and seal using colorless nail varnish. Wait the varnish dry out for 5 min. The slide can be analyzed under a fluorescence microscope immediately or stored at 4 °C up to one month. Figure 2 shows a scheme containing the main steps of this protocol section.


      Figure 2. Schematic diagram representing the main steps of the application of the protocol for CL Brener and Y strains (epimastigote forms). The halogenated thymidine analogs (CldU and IdU) are added separately in each culture for 12 h. After that, parasites must be washed with fresh media, mixed, and incubated for 24 h. Then samples must be washed, fixed, and added onto slides. Next, the parasite cells must have their DNA denatured and should be then processed for the detection of the thymidine analogs using primary antibodies (α-CldU and α-IdU) and the corresponding secondary antibodies (Alexa Fluor 488 α-rat and Alexa Fluor 568 α-mouse). Finally, mounting medium with DAPI must be added and the slides sealed. 

  2. Controls (checking the specificity of the antibodies)
    1. Prepare a new set of T. cruzi culture. Choose one strain if you carried out the previous analysis with two strains. Incubate T. cruzi cells (28 °C) until they reach exponential phase (~1 x 107 cells/ml) in 10 ml of culture. The parasites density varies according to the lineage used.
      Note: In our assays to check the specificity of the antibodies, we used only the T. cruzi Y strain in epimastigote forms.
    2. Follow the Steps A2-A5 exactly as previously described.
    3. Chill the 1x PBS to 4 °C.
      Note: Do not mix both cultures as previously done.
    4. After the incubation time, harvest separately the CldU- and IdU-incorporated cells by centrifugation at 800 x g for 5 min at
      4 °C and wash each sample twice using 5 ml of cold 1x PBS.
    5. Remove the 1x PBS from each sample carefully and suspend each pellet in 1 ml of cold FB. Transfer to a 1.5 ml microcentrifuge tube.
    6. Incubate each pellet at 4 °C for 7 min and wash them three times using 5 ml of cold 1x PBS (centrifuging at 800 x g for 5 min in each wash). For the last wash, suspend each pellet carefully into 500 μl of 1x PBS. If any of them is too small (i.e., almost invisible to the naked eye), decrease the volume to 100 μl of 1x PBS.
    7. Prepare four slides to receive the CldU-incorporated cells and four to receive the IdU-incorporated (totalizing eight slides). Spread 2.5 μl of PLS in each slide and let PLS dry out.
      Note: Use a coverslip to help spreading the PLS onto slide.
    8. Spread the suspended-pellet from each sample (i.e., CldU- and IdU-incorporated cells) (from Step B7) carefully in the slides (four slides for each thymidine analog group). Use 25 μl and save the remaining suspended-pellet volume in case you need to remake some slides.
      Note: Use the same surface where PLS was previous spread.
    9. Wait for the cells to settle on the slides for 10-15 min at room temperature. Ensure that the cells do not dry out.
    10. Permeabilize the cells by adding 50 μl of PS for 10 min at room temperature.
    11. Wash each slide containing cells three times using 1x PBS.
      Note: Use a P1000 micropipette to spread (by sneezing) 1x PBS (1 ml) onto slide three times.
    12. Denature the DNA of the cells by adding 50 μl of DB for 20 min at room temperature.
      Note: Use a plastic coverslip to help DB spread out and avoid dry out.
    13. Remove the plastic coverslip and wash each slide containing cells once using 1x PBS. 
    14. Neutralize the reaction by adding 50 μl of NB for 10 min at room temperature.
      Note: Use a plastic coverslip.
    15. Remove the plastic coverslip and wash each slide containing cells three times using 1x PBS. 
    16. Dilute both primary antibodies [i.e., rat α-CldU monoclonal antibody (Accurate) and mouse α-IdU monoclonal antibody (BD)] individually using two separated microtubes. Dilute each one 1:300 in BS.
    17. Spread out 50 μl of the diluted primary antibody rat α-CldU on the surface of two slides containing CldU-incorporated cells (from Step B15) and on the surface of two slides containing IdU-incorporated cells (from Step B15). Repeat the same procedure for the four remaining slides using the other diluted primary antibody, i.e., mouse α-IdU. Incubate for 2 h at room temperature.
      Note: Use a plastic coverslip to help primary antibodies spread out and avoid dry out. 
    18. Remove the plastic coverslip and wash the slide containing cells three times using 1x PBS.
    19. Dilute both secondary antibodies [i.e., Alexa Fluor 568-conjugated α-mouse (Thermo Scientific) and Alexa Fluor 488-conjugated α-rat (Thermo Scientific)] individually using two separated microtubes. Dilute each one 1:300 in BS.
      Note: From this step onwards, do not expose the slides to light.
    20. Spread out 50 μl of the diluted secondary antibody Alexa Fluor 488 α-rat on the surface of the following slides containing cells (from Step B19): CldU-incorporated + primary antibody rat α-CldU, CldU-incorporated + primary antibody mouse α-IdU, IdU-incorporated + primary antibody rat α-CldU, and IdU-incorporated + primary antibody mouse α-IdU. Repeat the same procedure for the four remaining slides (from Step B19) using the other diluted secondary antibody Alexa Fluor 568 α-mouse, i.e., CldU-incorporated + primary antibody rat α-CldU, CldU-incorporated + primary antibody mouse α-IdU, IdU-incorporated + primary antibody rat α-CldU, and IdU-incorporated + primary antibody mouse α-IdU. Incubate for 2 h at room temperature.
      Note: Use a plastic coverslip to help secondary antibodies spread out and avoid dry out. 
    21. Remove the plastic coverslip and wash each slide three times using 1x PBS.
    22. Ensure removing all the liquid from each and add 4 μl of Vectashield mounting medium containing DAPI.
      Note: This solution is used as the anti-fade mounting solution and to stain organelles containing DNA.
    23. Add a glass coverslip and seal each slide using colorless nail varnish. Wait the varnish dry out for 5 min. The slide can be analyzed under a fluorescence microscope immediately or stored at 4 °C up to one month. Figure 3 shows a scheme containing the main steps of this control protocol.


      Figure 3. Schematic diagram representing the main steps of antibodies specificity control. This control assay can be applied to any of the two previously strains (i.e., CL Brener and Y). The halogenated thymidine analogs (CldU and IdU) are added separately in each culture for 12 h. After that, each parasite-group (CldU-incorporated and IdU-incorporated) must be washed with 1x PBS, fixed, and aliquots distributed onto four slides (totalizing eight slides for the both groups). Next, each slide containing parasite cells must have their DNA denatured and should be then processed for the detection of the thymidine analogs using primary and secondary antibodies according to the specifications (on the scheme, see the specifications on the bottom of slides) (for details see previous Step B21). Finally, mounting medium with DAPI must be added in each slide and then sealed.

Data analysis

  1. Analyze each slide under a fluorescence microscope (see Equipment for specifications). Capture images using the differential interference contrast (DIC) (if available) or phase contrast. Also, capture images in the fields corresponding to the fluorescence emission blue (DAPI), green (CldU), and red (IdU). Capture images with the same exposure time, especially for CldU and IdU fields.
    Notes: 
    1. In our analysis, we used an objective lens 100x. Also, the exposure time used to capture the images can vary according to the microscope, filters, and software used. In our case, the images were captured using an exposure time ranging from 200-800 ms.
    2. Ensure a minimum of one hundred T. cruzi cells is captured per experiment. Each slide of the analysis of DNA exchange (Procedure A) is one technical replicate. This protocol must be prepared in biological triplicate from independent experiments. 
    3. To avoid bias, a double-blind approach should be applied, especially for paired analysis using two different strains. 
    4. In our analysis, we used the Olympus Cell F software (version 5.1.2640).
  2. Merge the images captured using ImageJ software (or other software that allows this approach). Figures 4 and 5 show representative images of cells containing, respectively: DNA exchange captured in each strain analyzed (i.e., CL Brener and Y), and antibodies specificity control.


    Figure 4. ADExTA applied in CL Brener and Y strains. Representative images are organized in six columns: DIC (to see morphology of the cells), DAPI (staining organelles containing DNA), CldU-incorporated cells (green), IdU-incorporated cells (red), merged 1 (CldU + IdU overlay), and merged 2 (DAPI + CldU + IdU overlay). The white arrows indicate cells that suffered DNA exchange, i.e., they have a nucleus containing CldU and IdU incorporated. Scale bars = 10 μm.


    Figure 5. Antibodies specificity control. Representative images of CL Brener strain show that the recognition of thymidine analogs is specific. CldU- and IdU-incorporated epimastigotes were added onto slides and processed for detection using mouse α-IdU + Alexa Fluor 568 (mouse), mouse α-IdU + Alexa Fluor 488 (rat), rat α-CldU + Alexa Fluor 568 (mouse), and rat α-CldU + Alexa Fluor 488 (rat). We can observe complete absence of cross-reactions between primary and secondary antibodies in each fluorescence channel analyzed (blue, red and green). Images were captured randomly. This figure was adapted from Alves et al. (2018). Scale bars = 10 μm.

  3. Count the number of total cells, cells CldU-labeled (green), cells IdU-labeled (red), cells CldU + IdU-labeled (yellow when merged), and cells non-labeled (blue-DAPI).
    Note: The number of total cells must be higher than one hundred to allow to obtain reliable results.
  4. Use the Excel software (Microsoft Office) to establish a table (or graphic) containing the percentage of cell CldU- and IdU-labeled, as well as double-labeled cells (i.e., CldU + IdU-labeled).
    Note: Other software can be used in place of Excel, for example, GraphPad Prism (GraphPad software).
  5. If you are comparing the DNA exchange between two different strains (e.g., CL Brener and Y strains), perform statistical analysis using Student’s t-test (two-tailed, unpaired t-test with Welch’s correction) to establish a P-value. Applying this test, you will find out if the difference observed is statistically significant or not. Figure 6 shows graphs with the percentage of DNA exchange (yellow column), as well as the P-value estimated for CL Brener and Y strains.


    Figure 6. Bar graphs showing the DNA exchange measurement. Bars represent the percentage of cells CldU-labeled (green), IdU-labeled (red), and CldU + IdU-labeled (yellow), which represents DNA exchange, for the two strains analyzed (Cl Brener and Y). More than 200 cells of each strain were analyzed per biological replicate. Error bars indicate SD of the triplicates. P-value was obtained using Student’s t-test. The numerical analysis presented in this figure was originally published in Alves et al. (2018).

Notes

  1. General notes
    1. This protocol should be carried out in biological triplicate from three independent experiments. Each experiment generates three technical replicates (three slides).
    2. Although the most companies that sell the halogenated thymidine analogs (BrdU, CldU or IdU) recommend a DNA denaturation step of 2 M HCl to allow their detection (e.g., Abcam, Sigma-Aldrich), we decided to use 2.5 M HCl to increase the number of sites containing the incorporated thymidine analogs. This approach improves the efficiency of detection, but impair DAPI staining, as evidenced in a recent study (da Silva et al., 2017a).

  2. Technical tips
    1. The incubation time of the thymidine analogs varies according to the doubling-time, and it can be optimized for other cell types. The same is valid for the incubation time after cell mix.
    2. Perform this protocol in the dark from the step that requires secondary antibodies incubation onwards.
    3. To avoid false positives, do not overexpose fluorescence images during capturing step.

Recipes

  1. Alkalinized water
    Prepare the alkalinized water by adjusting the pH of Milli-Q water to 9.5 (using 5 M NaOH with the aid of a pH meter)
  2. Phosphate buffered saline (1x PBS)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
    1. Prepare the buffer by adding 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4
    2. Add 800 ml of water and adjust the pH to 7.4 with HCl
    3. Complete the volume to 1 L
    4. Dispense the solution into aliquots (e.g., 250 ml) and sterilize by autoclaving (20 min, 120 °C, liquid cycle)
    5. Store at room temperature for up to six months
    6. Check the pH after prolonged use
  3. Liver Infusion Tryptose (LIT) medium
    68 mM NaCl
    5.3 mM KCl
    56 mM Na2HPO4
    0.2% (w/v) glucose
    0.5% (w/v) liver infusion broth
    0.5% (w/v) tryptose
    10 mg/L hemin
    10% (v/v) fetal bovine serum
    133 mg/L streptomycin sulfate salt
    59 mg/L penicillin G sodium salt
    1. Prepare the medium by adding 4 g of NaCl, 0.4 g of KCl, 8 g of Na2HPO4, 5 g of tryptose, 5 g of Liver infusion broth
    2. Add 800 ml of water, adjust pH to 7.3 (using 5 M NaOH with the aid of a pH meter), complete the volume to 880 ml, and sterilize by autoclaving (20 min, 120 °C, liquid cycle)
    3. Before utilization, add 1 ml of a hemin solution (10 mg/ml in 0.1 M of triethanolamine), 100 ml of fetal bovine serum, 20 ml of 20% (w/v) glucose, 133 mg of streptomycin sulfate, and 59 mg of penicillin
    4. Sterilize again by filtering (using 0.22 μm filter)
    5. Store at -20 °C for up to six months
  4. 5′-chloro-2′-deoxyuridine solution (CldU-S)
    10 mM of CldU diluted in water
    1. Prepare the buffer by adding 26.26 mg of 5′-chloro-2′-deoxyuridine in 9 ml of water
    2. Solubilizes and complete the volume to 10 ml
    3. Sterilize by filtering (using 0.22 μm syringe filter) and dispense the solution into aliquots (e.g., 1 ml) 
    4. Store at -20 °C for up to six months
  5. 5′-iodo-2′-deoxyuridine solution (IdU-S)
    10 mM of IdU diluted in water alkalinized
    Note: IdU is not readily soluble at physiological pH.
    1. Prepare the buffer by adding 35.4 mg of 5′-iodo-2′-deoxyuridine in 5 ml of alkalinized Milli-Q water (adjust the Milli-Q water pH to 9.5 using 5 M NaOH with the aid of a pH meter)
    2. Solubilize and complete the volume to 10 ml with Milli-Q water
    3. Sterilize by filtering (using 0.22 μm syringe filter) and dispense the solution into aliquots (e.g., 1 ml) 
    4. Store at -20 °C for up to six months
  6. Fixation buffer (FB)
    4% (w/v) Paraformaldehyde in 1x PBS
    1. Prepare the buffer by adding 2.1 g of Paraformaldehyde (powder, 95%) in 40 ml 1x PBS
    2. Solubilize and complete the volume to 50 ml with 1x PBS
    3. Store at 4 °C for up to one month
  7. Poly-L-lysine solution (PLS)
    0.1% (w/v) Poly-L-Lysine hydrochloride diluted in water
    1. Prepare the buffer by adding 10 mg of Poly-L-Lysine hydrochloride in 9 ml of water 
    2. Solubilize and complete the volume to 10 ml with water
    3. Sterilize by filtering (using 0.22 μm syringe filter) and dispense the solution into aliquots (e.g., 1 ml)
    4. Store at 4 °C for up to six months
  8. Permeabilization solution (PS)
    0.1% (v/v) Triton X-100 diluted in 1x PBS
    1. Prepare the buffer by adding 10 μl of Triton X-100 in 9,990 μl of 1x PBS
    2. Mix well, sterilize by filtering (using 0.22 μm syringe filter), and dispense the solution into aliquots (e.g., 1 ml)
    3. Store at 4 °C for up to six months
  9. Denaturation buffer (DB)
    2.5 M HCl
    1. Prepare the buffer by adding 2.1 ml of HCl (concentrated 37%) in 7.9 ml of water
    2. Mix carefully because this solution is irritant
    3. This solution cannot be stored and must be immediately made prior to use
  10. Neutralization buffer (NB)
    100 mM Boric Acid
    75 mM NaCl
    25 mM Sodium Tetraborate
    1. Prepare the buffer by adding 620 mg of Boric Acid, 440 mg of NaCl, and 950 mg of Sodium Tetraborate
    2. Add 80 ml of water and adjust the pH to 8.4 with NaOH
    3. Complete the volume to 100 ml and sterilize by filtering (using 0.22 μm syringe filter)
    4. Store at room temperature for up to six months
  11. Blocking solution (BS)
    4% (w/v) Bovine serum albumin dilute in 1x PBS
    1. Prepare the buffer by adding 400 mg of Bovine serum albumin (powder, ≥ 98%) in 9 ml 1x PBS
    2. Solubilize and complete the volume to 10 ml with 1x PBS
    3. Sterilize by filtering (using 0.22 μm syringe filter)
    4. Store at 4 °C for up to one month

Acknowledgments

We would like to thank all the members from the laboratories involved in developing this protocol. We also thank the support of the funding agencies: São Paulo Research Foundation (FAPESP) - Center of Toxins, Immune Response and Cell Signaling (CeTICS) (grants numbers 2013/07467-1, 2014/24170-5, 2017/18719-2), the National Council for Scientific and Technological Development (CNPq) (projects numbers: 870219/1997-9, 304329/2015-0), Minas Gerais Research Support Foundation (FAPEMIG), Coordination for the Improvement of Higher Education Personnel (CAPES), and The Wellcome Centre for Molecular Parasitology.

Competing interests

The authors declare that there is no conflict of interest regarding the publication of this article.

References

  1. Adl, S. M., Simpson, A. G., Lane, C. E., Lukeš, J., Bass, D., Bowser, S. S., Brown, M. W., Burki, F., Dunthorn, M., Hampl, V., Heiss, A., Hoppenrath, M., Lara, E., Le Gall, L., Lynn, D. H., McManus, H., Mitchell, E. A., Mozley-Stanridge, S. E., Parfrey, L. W., Pawlowski, J., Rueckert, S., Shadwick, L., Schoch, C. L., Smirnov, A. and Spiegel, F. W. (2012). The revised classification of eukaryotes. J Eukaryot Microbiol 59(5): 429-493. 
  2. Alves, C. L., Repolês, B. M., da Silva, M. S., Mendes, I. C., Marin, P. A., Aguiar, P. H. N., Santos, S. D. S., Franco, G. R., Macedo, A. M., Pena, S. D. J., Andrade, L. O., Guarneri, A. A., Tahara, E. B., Elias, M. C. and Machado, C. R. (2018). The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi. Sci Rep 8(1): 13335. 
  3. Browne, A. J., Guerra, C. A., Alves, R. V., da Costa, V. M., Wilson, A. L., Pigott, D. M., Hay, S. I., Lindsay, S. W., Golding, N. and Moyes, C. L. (2017). The contemporary distribution of Trypanosoma cruzi infection in humans, alternative hosts and vectors. Sci Data 4: 170050. 
  4. Calderano, S. G., Drosopoulos, W. C., Quaresma, M. M., Marques, C. A., Kosiyatrakul, S., McCulloch, R., Schildkraut, C. L. and Elias, M. C. (2015). Single molecule analysis of Trypanosoma brucei DNA replication dynamics. Nucleic Acids Res 43(5): 2655-2665. 
  5. da Silva, M. S., Cayres-Silva, G. R., Vitarelli, M. O., Marin, P. A., Hiraiwa, P. M., Araújo, C. B., Avila, A. R., Reis, M. S. and Elias, M. C. (2018). Transcription activity contributes to the firing of non-constitutive origins in Trypanosoma brucei. bioRxiv. doi: https://doi.org/10.1101/398016.
  6. da Silva, M. S., Monteiro, J. P., Nunes, V. S., Vasconcelos, E. J., Perez, A. M., Freitas-Junior Lde, H., Elias, M. C. and Cano, M. I. (2013). Leishmania amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation during cell cycle. PLoS One 8(11): e81397.
  7. da Silva, M. S., Muñoz, P. A. M., Armelin, H. A. and Elias, M. C. (2017a). Differences in the detection of BrdU/EdU incorporation assays alter the calculation for G1, S, and G2 phases of the cell cycle in trypanosomatids. J Eukaryot Microbiol 64(6): 756-770. 
  8. da Silva, M. S., Pavani, R. S., Damasceno, J. D., Marques, C. A., McCulloch, R., Tosi, L. R. O. and Elias, M. C. (2017b). Nuclear DNA replication in trypanosomatids: there are no easy methods for solving difficult problems. Trends Parasitol 33(11): 858-874. 
  9. Davidson, R. L. and Kaufman, E. R. (1979). Resistance to bromodeoxyuridine mutagenesis and toxicity in mammalian cells selected for resistance to hydroxyurea. Somatic Cell Genet 5(6): 873-885.
  10. Elias, M. C., da Cunha, J. P., de Faria, F. P., Mortara, R. A., Freymuller, E. and Schenkman, S. (2007). Morphological events during the Trypanosoma cruzi cell cycle. Protist 158(2): 147-157.
  11. Gaunt, M. W., Yeo, M., Frame, I. A., Stothard, J. R., Carrasco, H. J., Taylor, M. C., Mena, S. S., Veazey, P., Miles, G. A., Acosta, N., de Arias, A. R. and Miles, M. A. (2003). Mechanism of genetic exchange in American trypanosomes. Nature 421(6926): 936-939. 
  12. Hancock, A., Priester, C., Kidder, E. and Keith, J. R. (2009). Does 5-bromo-2'-deoxyuridine (BrdU) disrupt cell proliferation and neuronal maturation in the adult rat hippocampus in vivo? Behav Brain Res 199(2): 218-221.
  13. Herrick, J. and Bensimon, A. (1999). Single molecule analysis of DNA replication. Biochimie 81(8-9): 859-871. 
  14. Ponte-Sucre, A. (2016). An overview of Trypanosoma brucei infections: an intense host-parasite interaction. Front Microbiol 7: 2126.
  15. Stanojcic, S., Sollelis, L., Kuk, N., Crobu, L., Balard, Y., Schwob, E., Bastien, P., Pages, M. and Sterkers, Y. (2016). Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells. Sci Rep 6: 23142. 
  16. Torres-Guerrero, E., Quintanilla-Cedillo, M. R., Ruiz-Esmenjaud, J. and Arenas, R. (2017). Leishmaniasis: a review. F1000Res 6: 750.

简介

Trypanosoma cruzi 是属于锥虫科(Trypanosomatidae)的原生动物寄生虫。尽管锥虫病主要通过克隆生成繁殖,但多年来其中一些DNA交换的存在一直困扰着研究人员,主要是因为它可能代表了这些生物用来获得变异性的新形式。使用 T hymidine A nalogs(ADExTA)分析 D NA ex 变化是一种允许通过间接免疫荧光测定(IFA)以快速和简单的方式检测和测量DNA交换速率,特别是在锥虫细胞中的体外检测和测量速率的方法。该方法可用于通过配对分析检测一个锥虫病谱系内或不同谱系中的DNA交换。该测定的原理基于在DNA复制过程中掺入两种可区分的卤化胸苷类似物,称为5'-氯-2'-脱氧尿苷(CldU)和5'-碘-2'-脱氧尿苷(IdU)。将先前已与CldU和IdU分别掺入的两种细胞培养物混合后,可通过特异性抗体检测基因组中这些不寻常的脱氧核苷的存在。为此,需要DNA变性步骤以暴露掺入的胸苷类似物的位点。随后,使用荧光染料标记的抗体的二次反应将在荧光分析下产生不同的信号。通过使用该方法,使用标准荧光显微镜可以进行DNA交换验证(即,在同一细胞中存在CldU和IdU)。从胸苷类似物掺入到结果通常需要2-3天。值得注意的是,ADExTA相对便宜,不需要转染或严格的遗传操作。与需要DNA操作以在不同细胞中引入不同的药物抗性标记用于后部选择的其他耗时的方案相比,这些特征代表了优势。

【背景】Trypanosomatids寄生虫是Supergroup Excavata中的单细胞真核生物(Adl et al。,2012)。其中,有人类病原体负责引起几种破坏性疾病,如 T. cruzi (南美锥虫病的病原体,也称为美洲锥虫病), T. brucei (昏睡病的病原体,也称为非洲人类锥虫病)和 Leishmania spp。 (不同形式的利什曼病的病原体)。总而言之,这些奇特的生物每年造成超过50,000人死亡(Ponte-Sucre,2016; Browne et al。,2017; da Silva et al。,2017b; Torres-Guerrero et al。,2017)。虽然锥虫通过纵向二分裂主要通过克隆生成繁殖,但在这些生物中存在DNA交换,包括 T. cruzi 和 Leishmania spp。,多年来一直争论不休(Gaunt et al。,2003; Alves et al。, 2018)。由于缺乏实用和直接的实验,并且由于难以复制复杂的分析,DNA交换仍然很少被探索,尽管它可能代表了锥虫病用于获得变异性的新颖和未知形式。然而,我们小组最近开展的一项工作提出了一种检测不同 T菌株DNA交换的优雅方法。 cruzi (Alves et al。,2018)。在这项研究中,我们发现天然杂交CL Brener菌株(TcIV)相对于天然存在的非杂交Y菌株(TcII)的DNA交换率更高。此外,这项工作指出重组酶Rad51显着有助于该机制的效率(Alves et al。,2018)。

在这里,我们详细描述了本研究中用于检测锥虫病中DNA交换的方案。我们使用 T hymidine A将其命名为 D NA Ex 变化。 nalogs(ADExTA)。该方法的原理是基于两种可区分的卤化胸苷类似物[例如,5'-氯-2'-脱氧尿苷(CldU)和5'-碘-2'-脱氧尿苷(IdU)的掺入。 )]在细胞周期的S期进入DNA。在使用HCl的DNA变性步骤后,可以通过特异性抗体检测基因组中这些不寻常的脱氧核苷的存在,这将在使用荧光染料标记的抗体的二次反应后产生不同的信号。该原理与DNA复制的单分子分析技术相同(Herrick和Bensimon,1999),通常称为DNA梳理(Calderano et al。,2015; Stanojcic et al。 ,2016; da Silva et al。,2018)。该方法快速,相对简单,并且提供了DNA交换存在及其速率的良好概述。值得注意的是,与其他需要严格遗传操作并且耗时的繁重方法相比,该方案具有相当大的优势,例如使用重组DNA在不同细胞中引入不同的药物抗性标记的构建。通过使用该方法,可以使用标准荧光显微镜进行DNA交换的存在及其测量。理论上,该方案可以应用于允许掺入CldU / IdU的任何生物体,并且从胸苷类似物掺入到结果通常需要2-3天。值得注意的是,虽然胸苷类似物对某些细胞类型可能有毒(Davidson和Kaufman,1979; Hancock et al。等,2009),但在锥虫病中它们的使用显然没有任何毒性作用(Elias et al。,2007; da Silva et al。,2013; Stanojcic et al。,2016; da Silva et al。< / em>,2017a)。

关键字:DNA交换, 遗传交换, 胸苷类似物, CldU, IdU, DNA复制, 锥虫属, 克氏锥虫

材料和试剂

  1. 微管1.5 ml(Axygen,Maxyclear,目录号:MCT-150-C-S)
  2. 离心管15 ml(Corning,目录号:CLS430791)
  3. 离心管50 ml(Corning,目录号:CLS430290)
  4. 培养瓶25厘米 2 (康宁,斜颈,帽塞密封,目录号:CLS430168)
  5. 注射器过滤器0.22μm(Sartori,Minisart注射器过滤器,目录号:16534)
  6. 微量移液器吸头,10μl,200μl和1,000μl(Axygen,目录号:T-300,T-200-Y和T-1000-B)
  7. 血清移液器,10 ml(Costar Sterile,目录号:4488)
  8. 显微镜载玻片(Knittel玻璃,非彩色,目录号:VA111101FKB.01)
  9. 盖玻片(Knitell玻璃,22 x 22 mm,目录号:VD12222Y1A.01)
  10. 塑料盖玻片(使用切割塑料袋作为粘合剂)
  11. 吨。 cruzi 细胞系:Y株(TcII)和CL Brener株(TcIV)
  12. 5'-Iodo-2'-deoxyuridine(IdU)(Sigma-Aldrich,目录号:I7125)
  13. 5'-氯-2'-脱氧尿苷(CldU)(艾博抗(上海),目录号:ab213715)
  14. 小鼠α-IdU单克隆抗体(BD,目录号:347580)
  15. 大鼠α-CldU单克隆抗体(Accurate,目录号:YSRTMCA2060GA)
  16. 山羊α-小鼠IgG1二抗,Alexa Fluor 568(Thermo Scientific,目录号:A-21124)
  17. 山羊α-大鼠IgG(H + L)二抗,Alexa Fluor 488(Thermo Scientific,目录号:A-11006)
  18. 多聚甲醛(Sigma-Aldrich,目录号:158127)
  19. 聚L-赖氨酸盐酸盐(Sigma-Aldrich,目录号:P2658)
  20. 牛血清白蛋白(Sigma-Aldrich,目录号:05470)
  21. Triton X-100(Sigma-Aldrich,目录号:T8787)
  22. NaCl(Sigma-Aldrich,目录号:S9888)
  23. KCl(Sigma-Aldrich,目录号:P9541)
  24. Na 2 HPO 4 (Sigma-Aldrich,目录号:255793)
  25. KH 2 PO 4 (默克,目录号:104873)
  26. D-葡萄糖(Sigma-Aldrich,目录号:G8270)
  27. 肝脏输液汤(BD,目录号:226920)
  28. Tryptose(Sigma-Aldrich,目录号:70937)
  29. Hemin(Sigma-Aldrich,目录号:H9039)
  30. 三乙醇胺(Sigma-Aldrich,目录号:90279)
  31. 胎牛血清(Fisher Scientific,目录号:10500056)
  32. 青霉素G钠盐(Sigma-Aldrich,目录号:13752)
  33. 硫酸链霉素盐(Sigma-Aldrich,目录号:S9137)
  34. 硼酸(Sigma-Aldrich,目录号:B6768)
  35. 四硼酸钠(Sigma-Aldrich,目录号:221732)
  36. NaOH(默克,目录号:1064980500)
  37. 带DAPI的Vectashield安装介质(Vector Labs,目录号:H-1200)
  38. 盐酸发烟率37%(默克,目录号:100317)
  39. 指甲油(任何品牌,最好是无色)
  40. 碱化水(见食谱)
  41. 磷酸盐缓冲盐水(1x PBS)(见食谱)
  42. 肝脏输注胰蛋白酶(LIT)培养基(见食谱)
  43. 5'-氯-2'-脱氧尿苷溶液(CldU-S)(见食谱)
  44. 5'-碘-2'-脱氧尿苷溶液(IdU-S)(见食谱)
  45. 固定缓冲液(FB)(见食谱)
  46. 聚L-赖氨酸溶液(PLS)(见食谱)
  47. 透化解决方案(PS)(见食谱)
  48. 变性缓冲液(DB)(见食谱)
  49. 中和缓冲液(NB)(见食谱)
  50. 阻断解决方案(BS)(见食谱)

设备

  1. 微量离心机(Eppendorf,型号:5424 R)
  2. 电动移液器(Fisher Scientific,Fisherbrand,目录号:03-692-172)
  3. 水浴(Cientec,型号:CT-226)
  4. 孵化器BOD(Vitrex,型号:NI1705)
  5. 荧光显微镜[奥林巴斯,型号:BX51,耦合到XM10数码相机。滤波器规格:U-MWU2(激发= 330-385nm;发射= 420nm),U-MWIBA3(激发= 460-495nm;发射= 510-550nm)和U-MWG2(激发= 510-550nm) ;发射= 590 nm)]
  6. 微量吸管(Gilson,型号:Pipetman P10,P20,P200和P1000)
  7. 离心机(Eppendorf,型号:5810 R),配备4 x 250 ml摆动式转子
  8. 带盖玻片的Neubauer室(Sigma-Aldrich,型号:Bright-Line TM Hemacytometer)
  9. 生物安全II级A2柜(Pachane,型号:PA 700)
  10. pH计(Gehaka,型号:PG1800)
  11. 高压灭菌器

软件

  1. Olympus Cell F软件(Olympus,版本5.1.2640)
  2. ImageJ(NIH,版本1.47t)
  3. Microsoft Excel(Microsoft Office-任何版本)或GraphPad Prism(GraphPad软件公司)

程序

- 在开始使用此协议之前,我们建议您完全阅读它,尤其是本文末尾的“注释”部分。
- 细胞应在适合给定细胞类型的培养基和生长条件下培养。为了在数据分析期间获得合适数量的标记细胞,它们必须处于指数期。
- 虽然胸苷类似物在高剂量长时间使用时可能有毒,但我们没有发现我们的 T中有关毒性的任何改变。在我们的分析过程中,cruzi 细胞系。
- 重要提示:如果此方案中使用的抗体之前未进行特异性检测,我们强烈建议首先进行步骤B(检查抗体特异性的对照)。


  1. DNA交换分析
    1. 孵育 T. cruzi 细胞(28℃)直至它们在10ml培养物中达到指数期(~1×10 7 细胞/ ml)。寄生虫密度根据使用的谱系而变化。
      注意:在我们的分析中,我们使用了两种不同的谱系: T. cruzi Y菌株(TcII)和 T.克鲁兹 CL Brener菌株(TcIV),均为epimastigote形式。指数期可能因每种菌株而异。
    2. 拆分指数 T。将cruzi培养物加入两个培养瓶(25cm 2 )中,每瓶加入5ml。
    3. 在室温下解冻胸苷类似物CldU-S和IdU-S的两种溶液。
    4. 在一个烧瓶中加入一种胸苷类似物(例如,CldU)至终浓度为100μM,另一个烧瓶中加入另一个(例如,IdU),最终浓度为100μM。&nbsp;
    5. 将含有不同胸苷类似物的每个烧瓶在28℃孵育12小时。
      注意:孵育时间以及温度可能因每种寄生虫菌株或细胞类型而异。在我们的方案中,孵育时间(胸苷类似物掺入的时间)在经过长时间的测试后凭经验确定。我们设法使用相当于所用谱系倍增时间的一半的时间段获得了良好的结果( T. cruzi 倍增时间为24小时)。&nbsp;
    6. 在28℃温热等分试样的200ml肝输注胰蛋白酶(LIT)培养基。
      注意:如果可以,请使用水浴。
    7. 收集5毫升 T。 cruz 寄生虫CldU-或IdU-通过在800 x g 离心5分钟(来自步骤A6)而掺入。&nbsp;
    8. 小心地从细胞中取出培养基,并在28°C下用10 ml LIT替换。
    9. 小心地悬浮颗粒并重复步骤A7-A8两次,用LIT进行总共三次洗涤。对于最后一次洗涤,小心地将沉淀物悬浮于5ml LIT中。
    10. 混合使用CldU和IdU的 T。将cruzi 培养物放入一个烧瓶中,总体积为10ml。
    11. 在28°C孵育24小时。
      注意:孵育时间以及温度可能因每种寄生虫菌株或细胞类型而异。
    12. 将1x PBS冷却至4°C。
    13. 通过在4℃下以800×g离心5分钟收获细胞,并使用5ml冷的1x PBS洗涤两次。
    14. 小心地从细胞中取出1x PBS以保存沉淀。
    15. 将沉淀悬浮于1ml冷固定缓冲液(FB)中,并转移至1.5ml微量离心管中。
    16. 在4℃下孵育7分钟,并使用5ml冷的1x PBS(每次洗涤中800μl离心5分钟,每次洗涤5分钟)洗涤三次。对于最后一次洗涤,将沉淀小心地悬浮在500μl的1x PBS中。如果颗粒太小(即,肉眼几乎看不见),则将最后一次清洗的体积减少到100μl的1x PBS。
    17. 准备幻灯片以接收 T。通过将2.5μl聚-L-赖氨酸溶液(PLS)铺展到载玻片表面上直至PLS变干,克鲁兹细胞。为步骤A15中的每个样品准备三个载玻片。
      注意:使用盖玻片帮助将PLS传播到幻灯片上(有关详细信息,请参见图1)。


      图1.澄清步骤A17的插图。盖玻片可用于帮助将PLS液滴(2.5μl)分散到载玻片上。

    18. 在三个载玻片的每一个中小心地散布悬浮颗粒(来自步骤A16)。使用25-30μl并保存剩余的悬浮颗粒体积,以备您需要重新制作某些幻灯片时使用。
      注意:&nbsp;
      1. 使用PLS之前传播的相同表面。
      2. 每张幻灯片都是一个技术副本。
    19. 等待细胞沉淀并在室温下在载玻片上沉降10-15分钟。确保细胞不会变干。
    20. 通过在室温下加入50μl渗透固化溶液(PS)10分钟来使细胞透化。
    21. 使用1x PBS洗涤含有细胞的载玻片三次。
      注意:使用P1000微量移液管将1x PBS(1 ml)涂抹(滑动)至载玻片上三次。
    22. 通过在室温下加入50μl变性缓冲液(DB)20分钟使细胞DNA变性。
      注意:使用塑料盖玻片来帮助DB展开并避免干燥。
    23. 取出塑料盖玻片,用1x PBS清洗含有细胞的载玻片一次。&nbsp;
    24. 通过在室温下加入50μl中和缓冲液(NB)10分钟来中和反应。
      注意:使用塑料盖玻片来帮助NB展开。
    25. 取下塑料盖玻片,用1x PBS洗涤含有细胞的载玻片三次。&nbsp;
    26. 在封闭溶液(BS)中稀释一抗[即,大鼠α-CldU单克隆抗体(Accurate)和小鼠α-IdU单克隆抗体(BD)] 1:300。
    27. 在含有细胞的载玻片表面上展开50μl稀释的一抗(来自步骤A25)。在室温下孵育2小时。
      注意:使用塑料盖玻片来帮助一抗分散并避免干涸。&nbsp;
    28. 取下塑料盖玻片,用1x PBS洗涤含有细胞的载玻片三次。
    29. 在BS中稀释二抗[即,Alexa Fluor 568-缀合的山羊α-小鼠(Thermo Scientific)和Alexa Fluor 488-缀合的山羊α-大鼠(Thermo Scientific)] 1:300。
      注意:从此步骤开始,请勿将幻灯片曝光。
    30. 在含有载玻片的细胞上铺展50μl稀释的二抗(来自步骤A28)。在室温下孵育2小时。
      注意:使用塑料盖玻片来帮助一抗扩散并避免干涸。
    31. 取下塑料盖玻片,用1x PBS洗涤含有细胞的载玻片三次。
    32. 确保从含有细胞的载玻片表面除去所有液体,并加入4μl含有DAPI的Vectashield封固剂。
      注意:此解决方案用作抗褪色安装解决方案,用于染色含有DNA的细胞器。
    33. 添加玻璃盖玻片并使用无色指甲油密封。等待清漆干燥5分钟。可以在荧光显微镜下立即分析载玻片,或者在4℃下储存至一个月。图2显示了一个包含该协议部分主要步骤的方案。


      图2.表示应用CL Brener和Y菌株(epimastigote形式)方案的主要步骤的示意图。卤化胸苷类似物(CldU和IdU)在每种培养物中分别添加12 H。之后,必须用新鲜培养基洗涤寄生虫,混合并孵育24小时。然后必须洗涤,固定样品并将其添加到载玻片上。接下来,寄生虫细胞必须使其DNA变性,然后使用一抗(α-CldU和α-IdU)和相应的二抗(Alexa Fluor488α-大鼠和Alexa Fluor 568)进行处理以检测胸苷类似物。 α-小鼠)。最后,必须添加带有DAPI的封固介质并将载玻片密封。&nbsp;

  2. 对照(检查抗体的特异性)
    1. 准备一套新的 T。 cruzi 文化。如果您使用两个菌株进行先前的分析,请选择一个菌株。孵育 T. cruzi 细胞(28℃)直至它们在10ml培养物中达到指数期(~1×10 7 细胞/ ml)。寄生虫密度根据使用的谱系而变化。
      注意:在我们检测抗体特异性的分析中,我们只使用了 T. cruzi Y在epimastigote形式中的应变。
    2. 完全按照前面所述的步骤A2-A5进行操作。
    3. 将1x PBS冷却至4°C。
      注意:不要像以前那样混合两种文化。
    4. 孵育一段时间后,通过在800 x g 离心5分钟,分别收获掺入CldU和IdU的细胞。
      4℃,用5ml冷的1x PBS洗涤每个样品两次。
    5. 小心地从每个样品中取出1x PBS,并将每个沉淀悬浮在1ml冷FB中。转移至1.5 ml微量离心管中。
    6. 将每个沉淀在4℃孵育7分钟,并使用5ml冷的1x PBS洗涤它们三次(在每次洗涤中以800μL离心5分钟离心)。对于最后一次洗涤,将每个沉淀小心地悬浮在500μl的1x PBS中。如果它们中的任何一个太小(即,肉眼几乎看不见),将体积减少到100μl的1x PBS。
    7. 准备四个载玻片以接收掺入CldU的细胞,并制备四个用于接收IdU掺入的细胞(总计八个载玻片)。在每个载玻片中涂抹2.5μlPLS并让PLS变干。注意:使用盖玻片有助于将PLS扩散到载玻片上。
    8. 在载玻片中小心地从每个样品(即,CldU-和IdU-掺入的细胞)(来自步骤B7)扩散悬浮的沉淀物(每个胸苷类似物组的四个载玻片)。使用25μl并保存剩余的悬浮颗粒体积,以备您需要重新制作某些幻灯片时使用。
      注意:使用PLS之前传播的相同表面。
    9. 等待细胞在载玻片上在室温下沉降10-15分钟。确保细胞不会变干。
    10. 通过在室温下加入50μlPS10分钟使细胞透化。
    11. 使用1x PBS洗涤含有细胞的每个载玻片三次。
      注意:使用P1000微量移液管将1x PBS(1 ml)涂抹(滑动)至载玻片上三次。
    12. 通过在室温下加入50μlDB20分钟使细胞DNA变性。
      注意:使用塑料盖玻片来帮助DB展开并避免干燥。
    13. 取下塑料盖玻片,用1x PBS洗涤含有细胞的每个载玻片一次。&nbsp;
    14. 通过在室温下加入50μlNB10分钟来中和反应。
      注意:使用塑料盖玻片。
    15. 取下塑料盖玻片,用1x PBS洗涤含有细胞的每个载玻片三次。&nbsp;
    16. 使用两个分开的微管分别稀释一抗[即,大鼠α-CldU单克隆抗体(Accurate)和小鼠α-IdU单克隆抗体(BD)]。在BS中以1:300稀释每一个。
    17. 在含有掺入CldU的细胞(来自步骤B15)的两个载玻片的表面上和在含有掺入IdU的细胞的两个载玻片(来自步骤B15)的表面上展开50μl稀释的一抗大鼠α-CldU。使用另一种稀释的一抗即,小鼠α-IdU,对剩余的四个载玻片重复相同的步骤。在室温下孵育2小时。
      注意:使用塑料盖玻片来帮助一抗分散并避免干涸。&nbsp;
    18. 取下塑料盖玻片,用1x PBS洗涤含有细胞的载玻片三次。
    19. 使用两个分开的微管分别稀释二抗[即,Alexa Fluor 568-缀合的α-小鼠(Thermo Scientific)和Alexa Fluor 488-缀合的α-大鼠(Thermo Scientific)]。在BS中以1:300稀释每一个。
      注意:从此步骤开始,请勿将幻灯片曝光。
    20. 在含有细胞的下列载玻片(来自步骤B19)的表面上展开50μl稀释的二抗Alexa Fluor488α-大鼠:掺入CldU的+一抗大鼠α-CldU,掺入CldU的+一抗小鼠α- IdU,IdU掺入的+一抗大鼠α-CldU,和掺入IdU的+一抗小鼠α-IdU。使用其他稀释的二抗Alexa Fluor568α-小鼠, ie ,CldU掺入的+一抗大鼠α-CldU,CldU掺入对剩余的四个载玻片(来自步骤B19)重复相同的程序。 +一抗小鼠α-IdU,掺入IdU的+一抗大鼠α-CldU,和掺入IdU的一抗小鼠α-IdU。在室温下孵育2小时。
      注意:使用塑料盖玻片来帮助二抗扩散并避免干涸。&nbsp;
    21. 取下塑料盖玻片,用1x PBS清洗每个载玻片三次。
    22. 确保从每个液体中取出所有液体并加入4μl含有DAPI的Vectashield封固剂。
      注意:此解决方案用作抗褪色安装解决方案,用于染色含有DNA的细胞器。
    23. 添加玻璃盖玻片并使用无色指甲油密封每个载玻片。等待清漆干燥5分钟。可以在荧光显微镜下立即分析载玻片,或者在4℃下储存至一个月。图3显示了包含该控制协议主要步骤的方案。


      图3.表示抗体特异性控制的主要步骤的示意图。该对照测定可应用于两种先前菌株中的任何一种(,,CL Brener和Y)。在每种培养物中分别加入卤化胸苷类似物(CldU和IdU)12小时。之后,每个寄生虫组(掺入CldU和掺入IdU)必须用1x PBS洗涤,固定,并将等分试样分配到四个载玻片上(对于两组总共八个载玻片)。接下来,每个载有寄生虫细胞的载玻片必须使其DNA变性,然后根据规格使用一抗和二抗进行处理以检测胸苷类似物(在方案中,参见载玻片底部的规格)(详情见前面的步骤B21)。最后,必须在每个载玻片中加入含有DAPI的封固剂,然后密封。

数据分析

  1. 在荧光显微镜下分析每个载玻片(参见设备的规格)。使用差分干涉对比度(DIC)(如果可用)或相位对比度捕获图像。此外,在对应于荧光发射蓝(DAPI),绿色(CldU)和红色(IdU)的字段中捕获图像。使用相同的曝光时间捕获图像,尤其是CldU和IdU字段。
    注意:&nbsp;
    1. 在我们的分析中,我们使用了物镜100x。此外,用于捕获图像的曝光时间可根据所用的显微镜,滤光片和软件而变化。在我们的例子中,使用200-800毫秒的曝光时间捕获图像。
    2. 确保至少100个 T。每个实验捕获cruzi 细胞。 DNA交换分析的每张幻灯片(程序A)是一个技术复制品。该协议必须通过独立实验以生物学一式三份制备。
    3. 为避免偏见,应采用双盲方法,特别是对于使用两种不同菌株的配对分析。&nbsp;
    4. 在我们的分析中,我们使用了Olympus Cell F软件(版本5.1.2640)。
  2. 合并使用ImageJ软件(或允许此方法的其他软件)捕获的图像。图4和图5分别显示了含有细胞的代表性图像:分析的每个菌株中捕获的DNA交换(即,CL Brener和Y),以及抗体特异性控制。


    图4.在CL Brener和Y菌株中应用的ADExTA。 代表性图像分为六列:DIC(观察细胞形态),DAPI(含有DNA的染色细胞器),掺入CldU的细胞(绿色),掺入IdU的细胞(红色),合并1(CldU) + IdU覆盖),并合并2(DAPI + CldU + IdU覆盖)。白色箭头表示进行DNA交换的细胞,即,它们具有包含CldU和IdU的细胞核。比例尺=10μm。


    图5.抗体特异性控制。 CL Brener菌株的代表性图像显示胸苷类似物的识别是特异性的。将CldU-和IdU-掺入的epimastigotes添加到载玻片上并使用小鼠α-IdU + Alexa Fluor 568(小鼠),小鼠α-IdU + Alexa Fluor 488(大鼠),大鼠α-CldU + Alexa Fluor 568(小鼠)进行处理以进行检测。 )和大鼠α-CldU + Alexa Fluor 488(大鼠)。我们可以观察到在分析的每个荧光通道(蓝色,红色和绿色)中初级和二级抗体之间完全没有交叉反应。随机拍摄图像。该图改编自Alves et al。(2018)。比例尺=10μm。

  3. 计算总细胞数,细胞CldU标记的(绿色),细胞IdU标记的(红色),细胞CldU + IdU标记的(合并时为黄色)和未标记的细胞(蓝色-DAPI)。
    注意:总细胞数必须高于100,才能获得可靠的结果。
  4. 使用Excel软件(Microsoft Office)建立一个表(或图形),其中包含CldU和IdU标记的细胞百分比,以及双标细胞( ie ,CldU + IdU标记的)。
    注意:可以使用其他软件代替Excel,例如GraphPad Prism(GraphPad软件)。
  5. 如果您正在比较两种不同菌株(例如,CL Brener和Y菌株)之间的DNA交换,请使用Student's t -test进行统计分析(双尾,未配对t) - 使用Welch的校正测试)来建立 P - 值。应用此测试,您将发现观察到的差异是否具有统计显着性。图6显示了对于CL Brener和Y菌株估计的DNA交换百分比(黄色柱)以及 P 值的图表。


    图6.显示DNA交换测量的条形图。 条形图表示分析的两个菌株(Cl Brener和Y)的CldU标记(绿色),IdU标记(红色)和CldU + IdU标记(黄色)细胞的百分比,其代表DNA交换。 。每个生物学重复分析超过200个细胞的每个菌株。误差棒表示一式三份的SD。 P - 值是使用学生的 t - 测试获得的。该图中的数值分析最初发表在Alves et al。(2018)。

笔记

  1. 一般说明
    1. 该方案应在三个独立实验中以生物学一式三份进行。每个实验产生三个技术重复(三个幻灯片)。
    2. 虽然大多数销售卤化胸苷类似物(BrdU,CldU或IdU)的公司推荐使用2 M HCl的DNA变性步骤进行检测(例如,Abcam,Sigma-Aldrich),但我们决定使用2.5 M HCl增加含有掺入的胸苷类似物的位点数。这种方法提高了检测效率,但损害了DAPI染色,最近的一项研究证明了这一点(da Silva et al。,2017a)。

  2. 技术提示
    1. 胸苷类似物的孵育时间根据倍增时间而变化,并且可以针对其他细胞类型进行优化。这对于细胞混合后的孵育时间也是有效的。
    2. 从需要二抗孵育的步骤开始,在黑暗中执行此协议。
    3. 为避免误报,请勿在捕获步骤中过度曝光荧光图像。

食谱

  1. 碱化水
    通过将Milli-Q水的pH调节至9.5(使用5米NaOH,借助pH计)制备碱化水
  2. 磷酸盐缓冲盐水(1x PBS)
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    2mM KH 2 PO 4
    1. 通过添加8g NaCl,0.2g KCl,1.44g Na 2 HPO 4 和0.24g KH 2 来制备缓冲液。 PO <子> 4
    2. 加入800毫升水,用HCl调节pH至7.4
    3. 将音量调至1升
    4. 将溶液分配成等分试样(例如,250ml)并通过高压灭菌(20分钟,120℃,液体循环)灭菌
    5. 在室温下储存长达六个月
    6. 长时间使用后检查pH值
  3. 肝脏输注胰蛋白酶(LIT)培养基
    68 mM NaCl
    5.3 mM KCl
    56mM Na 2 HPO 4
    0.2%(w / v)葡萄糖
    0.5%(w / v)肝脏输液肉汤
    0.5%(w / v)胰蛋白胨 10毫克/升氯化血红素
    10%(v / v)胎牛血清
    133 mg / L硫酸链霉素盐
    59 mg / L青霉素G钠盐
    1. 通过加入4g NaCl,0.4g KCl,8g Na 2 HPO 4 ,5g胰蛋白胨,5g肝脏输注肉汤制备培养基
    2. 加入800毫升水,调节pH至7.3(使用5米NaOH,借助pH计),将体积调至880毫升,并通过高压灭菌消毒(20分钟,120℃,液体循环)
    3. 使用前,加入1 ml氯化血红素溶液(10 mg / ml,在0.1 M三乙醇胺中),100 ml胎牛血清,20 ml 20%(w / v)葡萄糖,133 mg硫酸链霉素和59 mg青霉素
    4. 通过过滤再次灭菌(使用0.22μm过滤器)
    5. 在-20°C下储存长达六个月
  4. 5'-氯-2'-脱氧尿苷溶液(CldU-S)
    在水中稀释的10 mM CldU
    1. 通过在9ml水中加入26.26mg 5'-氯-2'-脱氧尿苷制备缓冲液
    2. 溶解并完成10毫升的体积
    3. 通过过滤(使用0.22μm注射器过滤器)灭菌并将溶液分配成等分试样(例如,1ml)&nbsp;
    4. 在-20°C下储存长达六个月
  5. 5'-碘-2'-脱氧尿苷溶液(IdU-S)
    将10mM的IdU稀释于水中碱化 注意:IdU在生理pH下不易溶解。
    1. 通过在5ml碱化的Milli-Q水中加入35.4mg 5'-碘-2'-脱氧尿苷制备缓冲液(借助于pH计,使用5M NaOH将Milli-Q水的pH调节至9.5)
    2. 用Milli-Q水溶解并完成10 ml的体积
    3. 通过过滤(使用0.22μM注射器过滤器)灭菌并将溶液分配成等分试样(例如,1ml)&nbsp;
    4. 在-20°C下储存长达六个月
  6. 固定缓冲液(FB)
    1x PBS中4%(w / v)多聚甲醛
    1. 通过在40ml 1x PBS中加入2.1g多聚甲醛(粉末,95%)制备缓冲液
    2. 用1x PBS溶解并完成50ml的体积
    3. 在4°C下储存长达一个月
  7. 聚L-赖氨酸溶液(PLS)
    在水中稀释的0.1%(w / v)聚-L-赖氨酸盐酸盐
    1. 通过在9毫升水中加入10毫克聚-L-赖氨酸盐酸盐来制备缓冲液&nbsp;
    2. 溶解并用水将体积加至10ml
    3. 通过过滤灭菌(使用0.22μm注射器过滤器)并将溶液分配成等分试样(例如,1 ml)
    4. 在4°C下储存长达六个月
  8. 透化解决方案(PS)
    在1x PBS中稀释的0.1%(v / v)Triton X-100
    1. 通过在9,990μl的1x PBS中添加10μlTritonX-100来制备缓冲液
    2. 充分混合,过滤灭菌(使用0.22μm注射器过滤器),然后将溶液分配成等分试样(例如,1 ml)
    3. 在4°C下储存长达六个月
  9. 变性缓冲液(DB)
    2.5 M HCl
    1. 通过在7.9ml水中加入2.1ml HCl(浓缩37%)制备缓冲液
    2. 小心混合,因为这种溶液有刺激性
    3. 此解决方案无法存储,必须在使用前立即生成
  10. 中和缓冲液(NB)
    100 mM硼酸
    75 mM NaCl
    25 mM四硼酸钠
    1. 通过加入620毫克硼酸,440毫克NaCl和950毫克四硼酸钠来制备缓冲液
    2. 加入80毫升水,用NaOH调节pH至8.4
    3. 将体积减至100 ml并过滤灭菌(使用0.22μm注射器过滤器)
    4. 在室温下储存长达六个月
  11. 阻塞解决方案(BS)
    4%(w / v)牛血清白蛋白在1x PBS中稀释
    1. 通过在9ml 1x PBS中加入400mg牛血清白蛋白(粉末,≥98%)制备缓冲液
    2. 用1x PBS溶解并完成10ml的体积
    3. 通过过滤灭菌(使用0.22μm注射器过滤器)
    4. 在4°C下储存长达一个月

致谢

我们要感谢参与制定该议定书的实验室的所有成员。我们还感谢资助机构的支持:圣保罗研究基金会(FAPESP) - 毒素中心,免疫应答和细胞信号传导(CeTICS)(拨款号2013 / 07467-1,2014 / 24170-5,2017 / 18719-2 ),国家科学和技术发展委员会(CNPq)(项目编号:870219 / 1997-9,304329 / 2015-0),米纳斯吉拉斯州研究支持基金会(FAPEMIG),改善高等教育人员协调(CAPES)和Wellcome分子寄生虫学中心。

利益争夺

作者声明,本文的发布不存在任何利益冲突。

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引用:da Silva, M. S., Marin, P. A., Repolês, B. M., Elias, M. C. and Machado, C. R. (2018). Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi. Bio-protocol 8(24): e3125. DOI: 10.21769/BioProtoc.3125.
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