Imaging Assays to Detect DNA Damage in Trypanosome Parasites Using γH2A

Materials and reagents

Biological materials

1. Vero cells (ATCC CCL-81)

2. Trypanosoma cruzi CL Brener parasites expressing tdTomato (Tc) [12]

3. Bloodstream form (BSF) Trypanosoma brucei Plimmer and Bradford (ATCC PRA-382)

1. RPMI 1640 (HyClone, catalog number: SH30027.02)

2. 0.25% Trypsin-EDTA (Gibco, catalog number: 25200056)

3. Fibronectin powder (Corning, catalog number: 354008)

4. IMDM powder (Gibco, catalog number: 12200036)

5. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761)

6. Hypoxanthine (Sigma-Aldrich, catalog number: H9636)

7. Sodium hydroxide (Sigma-Aldrich, catalog number: S8045)

8. Sodium pyruvate (100 mM) (Gibco, catalog number: 11360070)

9. Thymidine (Sigma-Aldrich, catalog number: T1895)

10. L-cysteine (Sigma-Aldrich, catalog number: C7352)

11. Bathocuproine disulfonic acid (Sigma-Aldrich, catalog number: 146625)

12. Beta-mercaptoethanol (Millipore-Sigma, catalog number: M6250)

13. Heat-inactivated fetal bovine serum (FBS) (Gibco, catalog number: 10082147)

14. Penicillin-Streptomycin (Gibco, catalog number: 15070063)

15. Penicillin-Streptomycin-Glutamine (100×) (Gibco, catalog number: 10378016)

16. Phosphate buffered saline (PBS) (Gibco, catalog number: 20012027)

17. Paraformaldehyde 16% aqueous solution (Electron Microscopy Sciences, catalog number: 15710-S)

18. Triton X-100 (Sigma, catalog number: X100)

19. Tween-20 (Sigma, catalog number: P9416)

20. Bovine serum albumin (BSA) (Sigma, catalog number: A7906-500G)

21. Primary antibodies:

    1. Rabbit anti-T. cruzi γH2A polyclonal antibody (custom-made by Thermo Fischer Scientific)

       i. Epitope: KKARAT_pPSA

       ii. Bleed 3, affinity purified

    2. Rabbit anti-T. brucei γH2A polyclonal antibody (custom-made by Thermo Fischer Scientific)

       i. Epitope: KHAKAT_pPSV

       ii. Bleed 2, affinity purified

22. Secondary antibody: Goat anti-rabbit Alexa Fluor 488 (Abcam, catalog number: 150081)

23. Hoechst 33258 (Anaspec, catalog number: AS-83219)

24. VECTASHIELD Vibrance^® antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1800-2)

25. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650-100ML)

26. Phleomycin D1 (Zeocin) (Life Technologies, catalog number: R25001)

27. Compounds (as described in Rao et al. [5]):

    1. GNF6702 (kinetoplastid proteasome inhibitor)

    2. Benznidazole (Sigma-Aldrich, catalog number: 419656)

    3. CT0

    4. CT1

    5. CT3

Solutions

All solutions are stored at 4 °C. Solutions that are made fresh or need to be protected from light are indicated below each recipe.

1. RPMI complete medium (for Vero cells and T. cruzi parasites) (see Recipes)

2. 8% fixative solution (see Recipes)

3. 0.5% permeabilizing solution (see Recipes)

4. Blocking buffer 1 (see Recipes)

5. IFA wash buffer (see Recipes)

6. T. cruzi primary antibody solution (see Recipes)

7. T. cruzi secondary antibody solution (see Recipes)

8. Hoechst dye solution (see Recipes)

9. HMI-9 complete medium (for T. brucei) (see Recipes)

10. 4% fixative solution (see Recipes)

11. 0.25% permeabilizing solution (see Recipes)

12. Blocking buffer 2 (see Recipes)

13. T. brucei primary antibody solution (see Recipes)

14. T. brucei secondary antibody solution (see Recipes)

1. RPMI complete medium (for Vero cells and T. cruzi parasites)

   Reagent	Final concentration	Amount
   RPMI 1640	1×	1,000 mL
   FBS	9%	100 mL
   Penicillin-Streptomycin	0.9%	10 mL
   Total	n/a	1,110 mL

   Filter medium through a 0.22 µm, low-protein binding filter.




2. 8% fixative solution

   Reagent	Final concentration	Amount
   1× PBS	-	15 mL
   Paraformaldehyde (16%)	8%	15 mL
   Total	n/a	30 mL

   Make fresh on the day of the experiment.




3. 0.5% permeabilizing solution

   Reagent	Final concentration	Amount
   1× PBS	1×	500 mL
   Triton X-100	0.5%	2.5 mL
   Total	n/a	502.5 mL




4. Blocking buffer 1 (for T. cruzi)

   Reagent	Final concentration	Amount
   1× PBS	1×	100 mL
   BSA	4%	4 g
   Tween-20	0.1%	0.1 mL
   Total	n/a	100 mL




5. IFA wash buffer

   Reagent	Final concentration	Amount
   1× PBS	1×	1,000 mL
   Tween-20	0.1%	1 mL
   Total	n/a	1,000 mL




6. T. cruzi primary antibody solution

   Reagent	Final concentration	Amount
   Blocking buffer 1	1×	25 mL
   Primary antibody	1:250	0.1 mL
   Total	n/a	25 mL

   Make fresh on the day of the experiment.




7. T. cruzi secondary antibody solution

   Reagent	Final concentration	Amount
   Blocking buffer 1	1×	25 mL
   Secondary antibody	1:1,000	0.025 mL
   Total	n/a	25 mL

   Make fresh on the day of the experiment, protect from light.




8. Hoechst dye solution

   Reagent	Final concentration	Amount
   1× PBS	1×	25 mL
   BSA	1%	0.25 g
   Hoechst	1:250	0.1 mL
   Total	n/a	25 mL

   Make fresh on the day of the experiment, protect from light.




9. HMI-9 complete medium for T. brucei

   Reagent	Final concentration	Amount
   Water	n/a	730 mL
   IMDM powder	1×	1 packet
   Sodium bicarbonate	0.036 mM	3.02 g
   Hypoxanthine*	1.0 mM	0.136 g dissolved in 10 mL 0.1 M NaOH
   Sodium pyruvate	1.0 mM	10 mL
   Thymidine*	0.160 mM	0.039 g dissolved in 10 mL water
   L-cysteine*	1.0 mM	0.12 g dissolved in 10 mL water
   β-mercaptoethanol**	0.2 mM	14 µL diluted in 10 mL water
   Bathocuproine disulfonic acid*	0.050 mM	0.028 g dissolved in 10 mL water
   FBS	10%	100 mL
   Serum plus	10%	100 mL
   Penicillin-Streptomycin-Glutamine	1%	10 mL
   Total	n/a	1,000 mL

   *Solid salts are dissolved in 10 mL of water (or 0.1 M NaOH) before adding to medium.

   **β-mercaptoethanol is diluted in 10 mL of water before adding to medium. Combine ingredients and mix well. Filter medium through a 0.22 µm, low-protein binding filter.




10. 4% fixative solution

    Reagent	Final concentration	Amount
    1× PBS	-	12 mL
    Paraformaldehyde (16%)	4%	4 mL
    Total	n/a	16 mL

    Make fresh on the day of the experiment.




11. 0.25% permeabilizing solution

    Reagent	Final concentration	Amount
    1× PBS	1×	25 mL
    Triton X-100	0.25%	0.063 mL
    Total	n/a	25 mL




12. Blocking buffer 2 (for T. brucei)

    Reagent	Final concentration	Amount
    1× PBS	1×	100 mL
    BSA	3%	3 g
    Total	n/a	100 mL




13. T. brucei primary antibody solution

    Reagent	Final concentration	Amount
    Blocking buffer 2	1×	25 mL
    Primary antibody	1:250	0.1 mL
    Total	n/a	25 mL

    Make fresh on the day of the experiment.




14. T. brucei secondary antibody solution

    Reagent	Final concentration	Amount
    Blocking buffer 2	1×	25 mL
    Secondary antibody	1:1,000	0.025 mL
    Total	n/a	25 mL

    Make fresh on the day of the experiment, protect from light.

Laboratory supplies

1. 384-well black, clear and flat-bottom microplates (Greiner, catalog number: 781097)

2. Tissue culture–treated cell culture flasks with vented caps in T-25, T-75, T-175 sizes (Corning, catalog numbers: 430639, 430641U, and 431306, respectively)

3. Micropipettes of various sizes and compatible tips (e.g., Rainin, catalog number 30456871)

4. Neubauer Improved hemocytometer (SKC, Inc. INCYTO C-Chip^TM, catalog number: DHC-N015)

5. 0.22 µm vacuum filter and storage bottle (Corning, catalog number: 431205)

6. 15 mL and 50 mL conical tube (VWR, catalog numbers: 21008-216, 21008-242, respectively)

7. WypAll tissue wipes (WYPALL, catalog number: 34770)

8. 24-well plate (Corning, catalog number: 3524)

9. 1.7 mL microcentrifuge tube (Axygen, catalog number: MCT-175-C-S)

10. Poly-L-Lysine–coated coverslips, #1 thickness, 12 mm diameter (Corning, catalog number: 354085)

11. Parafilm (Amcor, catalog number: PM996)

12. Microscope slides (Propper, catalog number: 15400100)

13. Sharp-angled tweezers (Excelta, catalog number: 89411-808)

14. Blunt-end tweezers (Burkle, catalog number: 5386-0104)

15. KimTech Science KimWipes (Kimberley-Clark Professional, catalog number: 34120)

16. Immersion oil (Type F, Index = 1.518) (Nikon, catalog number: MXA22168)

17. Optional: Nalgene wash bottles (Thermo Fischer Scientific, catalog number: 2402-0500)

18. Optional: Benchtop vacuum aspirator

Equipment

1. Humidified incubator set at 37 °C, 5% CO₂

2. NucleoCounter (ChemoMetec, NC-200) and counting cassettes: Via1-Cassette (ChemoMetec, model: 941-0012)

3. Microplate reagent dispenser (Thermo Fisher Scientific Multidrop) and cassettes:

   1. Multidrop standard cassette (Thermo, catalog number: 24072670)

   2. Multidrop small tube cassette (Thermo, catalog number: 24073295)

4. Benchtop light microscope with 20× (0.3 NA) objective (Zeiss, Primovert, catalog number: 491206-0005-000)

5. Microplate orbital shaker (Perkin Elmer DELFIA PlateShake, catalog number: 1296-004)

6. Plate sealer (Agilent PlateLoc Thermal Microplate Sealer)

7. Fluorescence microscope

   1. ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices)

   2. Ti2-E epifluorescence microscope (Nikon)

Software and datasets

1. Nucleoview (ChemoMetec, version 1.2.0.0)

2. MetaXpress (Molecular Devices, LLC., version 6.7.0.211, 8 March 2021)

3. Prism v9.5.1 (GraphPad, January 26, 2023)

4. Excel (Microsoft, v2304, 25 April 2023)

5. NIS Elements AR (Nikon, version 5.20.02)

Procedure