Plasmodium cynomolgi Berok Growth Inhibition Assay by Thiol-reactive Probe Based Flow Cytometric Measurement

Materials and Reagents

1. 1.5 ml microcentrifuge tube (Axygen^®, catalog number: MCT-150-C)

2. 15 ml centrifuge tube (Corning, Costar, catalog number: CLS430052)

3. 50 ml centrifuge tube (Corning, Costar, catalog number: CLS430291)

4. 24-well flat-bottom plates (Corning, Costar, catalog number: CLS3738)

5. 96-well round-bottom plates (Corning, Costar, catalog number: CLS3367)

6. 96-well flat-bottom plates (Corning, Costar, catalog number: CLS3370)

7. Non-vented T75 flasks (Corning, Costar, catalog number: CLS3814)

8. 3 ml round bottom polystyrene tubes (Becton Dickinson, catalog number: 156758)

9. Sterile V-channel reservoirs (VWR, catalog number: VWRI613-1174)

10. Sterile 14 mm Petri dish (VWR, catalog number: VWRI390-1373)

11. Non-woven filters (Antoshin)

12. Aluminium foil

13. Lithium heparin vacuteiners (Becton Dickinson, catalog number: 367880)

14. Clot activator vacuteiners (Becton Dickinson, catalog number: 367820)

15. P. cynomolgi Berok erythrocyte culture (Bruce Russell, University of Otago) (Chua et al., 2019)

16. Macaca fascicularis erythrocytes (Monash Animal Research Platform, Monash University)

17. M. fascicularis serum (Monash Animal Research Platform, Monash University)

18. Pre-immune and P. vivax antibodies (Yang Cheng, Jiangnan University) (Shen et al., 2021)

19. RPMI 1640 medium supplemented with GlutaMAX (Gibco, catalog number: 61870-036)

20. HEPES (Sigma-Aldrich, catalog number: H4034)

21. D-glucose (Sigma-Aldrich, catalog number: G7021)

22. Hypoxanthine (Calbiochem, catalog number: 4010BC)

23. PBS tablets (Gibco, catalog number: 18912014)

24. Giemsa (VWR, catalog number: 352603R)

25. Methanol (Fisher Scientific, catalog number: A412-4)

26. Hoechst (Sigma-Aldrich, catalog number: 63493)

27. Mitotracker Deep Red (Thermo Fisher Scientific, FM Molecular Probes, catalog number: 22426)

28. Complete culture media (see Recipes)

Equipment

1. Class II Microbiological Safety Cabinet

2. Microplate orbital shaker

3. Multichannel pipette (12-Channel pipette, 10-100 μl) (Thermo Scientific^TM, catalog number: 4661070N)

4. Benchtop plate centrifuge (Eppendorf, model: 5804R)

5. Light microscope with 100× oil immersion lens (NIKON, model: YS100)

6. Canto II flow cytometer (Becton Dickinson, Canto II)

7. Tri-mix gas with 5% O₂, 5% CO₂, and the rest with N₂

8. Hypoxia incubator chamber (Stemcell, catalog number: 27310)

9. 37°C incubator

10. 4°C fridge

11. -20°C freezer

Software

1. FACSDiva 6.1.3 software (Becton Dickinson)

2. Flowjo, LLC (Becton Dickinson, Tree Star Inc, https://www.flowjo.com/contact)

3. Microsoft Excel (Microsoft, https://www.microsoft.com/en-us/microsoft-365/excel)

4. Prism 8 (GraphPad, https://www.graphpad.com/scientific-software/prism/)

Procedure

1. Parasite culture

   1. Thaw a frozen vial containing 1 ml of P. cynomolgi Berok parasitized erythrocytes using a series of sodium chloride solutions in a dropwise manner, according to Moll et al. (2008).

   2. Resuspend the pelleted parasitized erythrocytes in complete culture media in a 24-well plate and incubate the parasite culture at 5% hematocrit in a Hypoxia incubator chamber filled with tri-mix gas in a 37°C incubator.

   3. After overnight incubation, check for successful parasite maturation before expanding the parasitized erythrocytes in a non-vented flask in a 25-cm² cell culture flask at 5% hematocrit using M. fascicularis erythrocytes, according to Chua et al. (2019).

   4. If parasite culture shows a mix of early trophozoites and schizont stage (1:1) parasitized erythrocytes 2 days before the experiment day, perform a “quick and dirty” culture synchronisation.




2. “Quick and dirty” culture synchronisation

   1. About 30 h before the experiment, pellet the parasite culture (at least 2 ml culture at 5% hematocrit) at 1,114 × g for 2 min at room temperature.

   2. Set aside the supernatant (complete culture media) in a separate 50 ml Falcon tube.

   3. Carefully aspirate and remove the brownish-black coloured top layer of pelleted erythrocytes (schizonts that have not egressed) manually with a pipette.

   4. Using a fresh pipette tip, gently resuspend the pelleted red blood cells with the supernatant set aside previously, and transfer the mixture into a non-vented T75 flask. Gas with tri-mix gas for 5 s, and incubate flask at 37°C overnight.

   5. Check that the overnight parasitized erythrocytes have matured to the schizont stage (at least 70%) the following afternoon.




3. Antibody preparation for GIA

   1. Thaw frozen vials of antibodies on ice, and prepare fresh after confirmation of schizont maturation in parasitized erythrocyte culture.

   2. In the antibody dilution plate, fill all outer wells of the 96-well round-bottomed plate with 200 μl of sterile water.

      Note: If a flat-well bottomed plate is used, place the plate on an angle to ensure consistent pipetting at the bottom of each well.

   3. Label the lid of the plate to indicate rows with the antibody name and columns from “1” to “10”.

   4. Seed wells labelled “2” to “10” with 20 μl of complete media (see Figure 1A for plate layout).

   5. Dilute the corresponding antibodies to their respective wells labelled with “1” with a final volume of 30 μl at 1 mg/ml with culture media.

   6. Perform a threefold serial dilution by transferring 10 μl from well “1” to well “2”. Mix by pipetting.

   7. Repeat the serial dilution of antibodies up to well “10”.

      
      

      
      Figure 1. Antibody and experimental plates layout. The blue coloured wells show wells filled with 200 μl sterile water. Blanks are indicated as ‘X’. A. An illustration of the antibody plate layout with labels indicating antibodies from Rows A to H. Columns 1 to 12 indicate the decreasing antibody concentration. Orange coloured wells indicate 20 μl complete media (CM) as antibody diluent. B. An illustration of the experimental plate layout with labels indicating antibodies from Rows A to H and concentrations from 1 to 10. A positive control is included in Row B, with a negative control in Row G. Dark red coloured wells indicate wells containing infected red blood cells (iRBC), and the light red coloured wells indicate uninfected red blood cells (uRBC).

      
      

4. P. cynomolgi Berok GIA – Set up

   1. Prior to set up, ensure that the majority of parasitized erythrocytes are in the mid-late schizont stage (at least 70% of parasitized erythrocytes in culture, with each schizont containing at least 8 merozoites).

   2. To minimise media evaporation, fill all outer wells of the 96-well round-bottomed or flat-bottomed plate with 200 μl of sterile water, and place a covered 14 mm Petri dish containing sterile water in the hypoxia incubator chamber.

   3. Label the lid of the plate to indicate in duplicate the antibody name and their corresponding test dilution with wells “1” to “10”. Include a row each of positive and negative control wells (see Figure 1B for plate layout).

   4. Seed negative control wells with 70 μl of uninfected red blood cells at 1% hematocrit.

   5. Seed positive control wells with 70 μl of parasitized red blood cells at 1% hematocrit and 0.5% parasitaemia.

   6. The final volume for each test well is 70 μl. Calculate the volume of pelleted erythrocytes for a final volume of 70 μl/well at 1% hematocrit and 0.5% parasitaemia. However, as antibodies will be diluted 10× to attain a final concentration of 100 μg/ml, resuspend pelleted erythrocytes with complete culture media volume at 63 μl/well instead.

   7. Transfer the mixture into a sterile V-channel reservoir.

   8. Mix the solution in the reservoir by gently pipetting up and down to ensure a homogenous solution before pipetting 63 μl of the parasite culture into each test well.

   9. Add 7 μl of antibodies (from Step C6) to each well in duplicate using a multi-channel pipette.

   10. Incubate the plates in the hypoxia incubator chamber flushed with tri-mix gas at 37°C.

   11. To maintain a healthy parasitized erythrocyte culture at the second cycle of merozoite invasion in this 2 cycle assay, add 7 μl of complete culture media (with freshly supplemented serum) to each well at 48 h post incubation.




5. P. cynomolgi Berok GIA – Staining

   1. At 96 h post incubation, gently resuspend the parasite culture before extracting 20 μl into a new 96-well round-bottomed plate for staining.

   2. Pellet red blood cells by spinning the plate in a centrifuge at 400 × g for 5 min at room temperature with low deceleration.

   3. Prepare a staining MasterMix for a final volume of 50 μl/well with 8 μM Hoechst 34580 and 150 nM Mitotracker Deep Red FM in 1× PBS.

   4. After the spin is complete, remove as much supernatant as possible from each well.

   5. Resuspend the pelleted red blood cells with 50 μl of staining MasterMix.

   6. Incubate the plate on the microplate orbital shaker at 120 rpm for 20 min at room temperature. To prevent quenching of the dye, cover the shaker with a big sheet of aluminium foil (reusable).

   7. Pellet red blood cells by spinning the plate in a centrifuge at 400 × g for 5 min at room temperature with low deceleration.

   8. Remove as much supernatant as possible, and wash cells with 200 μl of PBS.

   9. Repeat the centrifugation and washing steps twice.

   10. Resuspend cells in 200 μl of PBS, and transfer mixture to a 3 ml round bottom polystyrene tube for analysis on the flow cytometer.




6. Analysis of Hoechst 34580⁺/Mitotracker Deep Red FM⁺ parasitized erythrocytes by flow cytometry

   1. Measure parasitaemia using FACS.

   2. Record 50,000 to 100,000 cells for each sample.

   3. Export data to fsc file format.

Data analysis

1. Using Flowjo software, gate single cells (x axis: SSC-H, y axis: FSC-H), then select the corresponding detector channels based on the single-cell population and gate the double-positive Hoechst 34580⁺/Mitotracker Deep Red⁺ population.

2. Export gated population values to Excel software.

3. Subtract the baseline value (negative control) from test samples.

4. Copy values from Excel software to GraphPad Prism 8 software.

5. To compare the percentage of Hoechst 34580⁺/Mitotracker Deep Red⁺ viable parasites across different drug concentrations, drug concentrations are first log₂ transformed and the data values are then normalized.

6. Use the four-parameter logistic model or log (inhibitor) vs. response – variable slope function for dose-response curve fitting.

7. The mean IC₅₀ values for various antibodies are compared using one-way ANOVA with the Tukey post hoc test function.

Recipes

1. Complete culture media

   RPMI-1640 medium, GlutaMAX^TM supplement, HEPES with the following additions:

   2.5 ml HEPES (1 M)

   1.0 g/L D-(+)-Glucose

   0.05 g/L Hypoxanthine

   20% (vol/vol) M. fascicularis serum

   Sterile filter and store at 4°C

Acknowledgments

This work was supported by the Research Grant from the Institute of Medical Sciences, Kangwon National University 2020 (J-H. H), and by the Marsden Fund 17-U00-241 from the B.R. laboratory (B. R.). The protocol is adapted from Shen et al. (2021).

Competing interests

The authors declare no competing financial interests.

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