Isolation of GFP-expressing Malarial Hypnozoites by Flow Cytometry Cell Sorting

Materials and Reagents

1. Collagen-coated 96-well plates (PerkinElmer, catalog number: 6055700)

2. Cellstar 50 ml polypropylene tubes (Greiner Bio-One, catalog number: 227261)

3. 5 ml serological pipettes (for example, VWR, catalog number: 612-5523)

4. 10 ml serological pipettes (for example, Greiner Bio-One, catalog number: 607-107)

5. 25 ml serological pipettes (for example, VWR, catalog number: 612-5544)

6. 1.5 ml Sarstedt tubes, sterile (Sarstedt, catalog number: 72.692.405)

7. 5 ml round-bottomed polystyrene test tubes (Falcon, catalog number: 352052)

8. 5 ml round-bottomed polystyrene test tubes, with cell strainer snap cap (Falcon, catalog number: 352235)

9. Reagent reservoir (Greiner Bio-One, catalog number: 960305)

10. Pipette tips 300 µl and 1,000 µl (Rainin, SR LTS filter tip 768)

11. Phosphate-buffered saline (PBS, 1×) (Thermo Fisher Scientific, catalog number: 14200-067)

12. 0.25% trypsin/EDTA (Thermo Fisher Scientific, catalog number: 25200072)

13. TRIzol reagent (Thermo Fisher Scientific, catalog number: 15596026)

14. William’s E medium + Glutamax (Thermo Fisher Scientific, catalog number: 32551-087)

15. Human serum A+ (pooled, heat inactivated; Sanquin blood bank)

16. Insulin/transferrin/selenium supplement 100× (Thermo Fisher Scientific, catalog number: 41400-045)

17. Sodium pyruvate 100 mM (Thermo Fisher Scientific, catalog number: 11360-036)

18. MEM-NEAA 100× (Thermo Fisher Scientific, catalog number: 11140-035)

19. Pen/strep 100× (Thermo Fisher Scientific, catalog number: 15140-122)

20. Hydrocortisone (Sigma, catalog number: H0888)

21. 50 mM β-mercaptoethanol (Thermo Fisher Scientific, catalog number: 31350-010)

22. DMSO hybrimax (Sigma, catalog number: P1860)

23. William’s B medium (see Recipes)

24. 20% William’s B medium (see Recipes)

25. 0.1 M hydrocortisone (see Recipes)

Primary cells and parasites

1. Primary macacque hepatocytes (freshly isolated or thawed from a cryopreserved stock; Macaca mulatta or Macaca fascicularis; source: BPRC; hepatocytes may be obtained through commercial suppliers

2. GFP-expressing P. cynomolgi lines as described in Voorberg-van der Wel et al. (2013 and 2020); lines can be obtained upon request from the corresponding author

Equipment

1. (Multichannel) pipettes (Rainin)

2. Pipette controller (Integra Biosciences, model: Pipetboy acu 2)

3. Refrigerated benchtop centrifuge (Beckman Coulter, model: Allegra X-15R)

4. Inverted microscope (Leica, model: DMi1)

5. Laminar flow biosafety cabinet IIB (Clean-Air EF/B6)

6. Fume hood

7. Humidified 5% CO₂ incubator at 37°C

8. Water bath at 37°C

9. Ice machine

10. Vortex

11. High Content Imager – Operetta (PerkinElmer)

12. Cell sorter – FACS Aria I flow cytometer (BD Biosciences) equipped with a blue laser (20 mW)

13. -80°C freezer (Eppendorf)

Software

1. FlowJo software version 9.9 (https://www.flowjo.com/solutions/flowjo/downloads/previous-versions)

2. FACS Diva version 8.0.1 (https://www.bdbiosciences.com/en-us/instruments/research-instruments/research-software/flow-cytometry-acquisition/facsdiva-software)

3. Columbus 2.8.2 (PerkinElmer) (https://www.perkinelmer.com/nl/product/image-data-storage-and-analysis-system-columbus)

Procedure

The different steps of the protocol are depicted in Figure 1 and described in detail below.

Figure 1. Overview of the procedure. Briefly, primary macaque hepatocytes are plated into collagen-coated 96-well plates, and after 2-3 days are inoculated with GFP-expressing P. cynomolgi sporozoites isolated from A. stephensi mosquitoes. At 6 days post-inoculation the culture is imaged using an Operetta high content imager to assess the presence of hypnozoites and schizonts. The cells are then detached by trypsinization and this pooled material is used for flow cytometry cell sorting of hypnozoites and schizonts.

1. Infection and culture of macaque primary hepatocytes with transgenic P. cynomolgi parasites

   1. Plate 65 × 10³ macaque primary hepatocytes per well (96-well plates; see Note 2) and maintain the culture at 5% CO₂ and 37°C in William’s B medium (100 µl per well; see Recipe 1) supplemented with 2% DMSO. Hepatocytes can be freshly isolated or used from a cryopreserved stock. After 2-3 days, inoculate the cultures with 50 × 10³ fluorescent transgenic P. cynomolgi sporozoites per well (Voorberg-van der Wel et al., 2013 ) as described in https://bio-protocol.org/e3722.

   2. Maintain the culture in William’s B medium without DMSO (this affects parasite development) at 5% CO₂ and 37°C; refresh the medium three times per week by removing as much culture medium as possible, followed by gently adding fresh William’s B medium (100 µl per well). Routinely, the culture is maintained for 6 days because at that time, hypnozoites and developing liver stages can be distinguished based on their size.

   3. Culture alongside uninfected wells with macaque primary hepatocytes as the control (≥6 wells of a 96-well plate).




2. Harvesting cells

   1. On the day of harvest, check the quality of infection (for reference) by live imaging on a fluorescence microscope/High Content Imager (Voorberg-van der Wel et al., 2013 ). Please see the image in the flowchart for an example. Further insights into liver-stage parasite development can be gained from Voorberg-van der Wel et al. (2020) .

   2. Remove the supernatant from the 96-well plates and wash with 1× in PBS (100 µl/well).

   3. Remove the supernatant and add 30-50 µl pre-warmed (at 37°C) 0.25% trypsin/EDTA per well; incubate for 3 min at 37°C until the cells have detached (check with an inverted microscope).

   4. Add 100 µl William’s B medium per well to stop trypsinization.

   5. Mix by gently pipetting up and down (using a multichannel pipette) and pool the contents of the wells (containing the detached cells and fluids) into a reagent reservoir.

   6. Transfer the material into a 50-ml tube and spin (2 min 200 × g, RT, brake medium).

   7. Wash twice in 20% William’s B medium (each time spin 2 min 200 × g, RT, brake medium).

   8. Pass the sample, while gently pipetting up and down, through a cell strainer snap cap (on top of 5-ml round-bottomed polystyrene test tubes) to exclude clumps.

   9. Keep cells on ice until sorting.




3. Fluorescence-activated cell sorting

   1. Pre-cool the sample loader at 4°C and rotate at 300 rpm during cell sorting.

   2. Set up the FACSAria according to the manufacturer’s procedures, e.g., run Cytometer Setup and Tracking beads (CS&T).

   3. Install a 100-µm nozzle and set the sheath pressure to 20 psi (default setting).

   4. Record 100,000 events of the uninfected hepatocyte culture as well as the transgenic liver-stage parasite culture to check the gating settings.

   5. Insert 1.5-ml collection tubes filled with 300 µl TRIzol if the material is to be used for transcriptomics

      NB: TRIzol is toxic: fill the tubes in the fume hood and dispose of the waste separately.

   6. Sort (sort precision: 4-way purity mode) the fractions of interest (Figure 2).

      
      

      
      Figure 2. Gating strategy for sorting hypnozoites and schizonts on day 6. Parasite populations are gated based on FITC expression. The GFPlow gate contains hypnozoites and the GFPhigh gate contains developing forms (schizonts). The GFPdim gate contains a mixture of parasites.

   
   

   7. When approximately half of the sample has been sorted, sort a small amount of sample into 100 µl 20% William’s B (collect about 100 GFPlow and GFPhigh events each; also take a sample of the other fractions at the same time) and transfer to a 96-well plate for a quality check of the Operetta system (Voorberg-van der Wel et al., 2013 ).

   8. When the sort has finished, vortex the TRIzol tubes for 30 s and transfer to the -80°C freezer.

Notes

1. Please note that working with the materials listed above potentially involves biological risks. Monkey-derived hepatocytes should be treated as biohazard material and may contain infectious agents. P. cynomolgi parasites are infectious to humans. All handling of these materials should be performed under BSL2 conditions.

2. While the original work was performed in 6-well plates, we have noticed that infection grades in 96-well plates are higher.

3. Instead of using uninfected hepatocytes as the negative control, macaque primary hepatocytes infected with P. cynomolgi wild-type parasites may be used (Voorberg-van der Wel et al., 2013 ).

4. Hepatocytes are highly autofluorescent; therefore, it is difficult to distinguish between uninfected cells and those containing GFPlow-expressing parasites (hypnozoites). We have empirically determined that using the PE channel in combination with the FITC channel provides an efficient method to separate the different parasitic populations.

5. Implementing a doublet and debris exclusion through the addition of a scatterplot of FSC versus SSC, followed by a scatterplot of FSC-A versus FSC-H, may be beneficial for removing subcellular debris and multi-cell aggregates.

6. Instead of harvesting all the wells, it is recommended to perform live imaging and fix a few wells for IFA (https://bio-protocol.org/e3722) to establish the infection characteristics.

7. Co-staining with Sytox Blue can be included as a live/dead indicator, as described by others ( Posfai et al., 2018 ). Alternatively, a surface stain found on all intact hepatocytes could potentially be used in combination with, for example, PE-Cy7.

8. Depending on the cell sorting apparatus, a 130-µm nozzle can be used as an alternative to a 100-µm nozzle. This may be beneficial for maintaining host cell integrity.

Recipes

1. William’s B medium

   William’s E medium with glutamax

   10% human serum (A+)

   1% MEM nonessential amino acids (NEAA)

   2% penicillin/streptomycin

   1% insulin/transferrin/selenium

   1% sodium pyruvate

   50 µM β-mercaptoethanol

   0.05 µM hydrocortisone

   
   

   1. 500 ml William’s E medium

   2. 50 ml Human Serum A+

   3. 5 ml 100× pen/strep (10,000 U/ml)

   4. 5 ml 100× insulin/transferin/selenium supplement

   5. 5 ml 100 mM sodium pyruvate

   6. 5 ml 100× MEM-NEAA

   7. 250 µl 0.1 M hydrocortisone

   8. 500 µl 50 mM β-mercaptoethanol

   9. Store at 4°C and use within 1 month

2. 20% William’s B medium

   Dilute William’s B medium 1:5 with William’s E medium

3. 0.1 M hydrocortisone

   1. Dissolve 50 mg hydrocortisone in 1 ml DMSO

   2. Store 250-µl aliquots at 4°C

Acknowledgments

This work was supported by a grant from the Medicines for Malaria Venture, a translational research grant (WT078285) from the Wellcome Trust, FP7 EU grants MALSIG (contract number 223044) and EVIMALAR (contract number 242095), and the Bill and Melinda Gates foundation (OPP1141292).

Ethics

Nonhuman primates were used because no other models (in vitro or in vivo) were suitable for the aims of this project. The local independent ethics committee conforming to Dutch law (BPRC Dier Experimenten Commissie, DEC) approved the research protocol (agreement number DEC# 708) prior to the beginning of the study, and the experiments were performed according to Dutch and European laws. Further details can be found in the original publication (Voorberg-van der Wel et al., 2017).

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

References

1. Bertschi, N. L., Voorberg-van der Wel, A., Zeeman, A. M., Schuierer, S., Nigsch, F., Carbone, W., Knehr, J., Gupta, D. K., Hofman, S. O., van der Werff, N., Nieuwenhuis, I., Klooster, E., Faber, B. W., Flannery, E. L., Mikolajczak, S. A., Chuenchob, V., Shrestha, B., Beibel, M., Bouwmeester, T., Kangwanrangsan, N., Sattabongkot, J., Diagana, T. T., Kocken, C. H. and Roma, G. (2018). Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite Plasmodium cynomolgi during progression into dormancy. Elife 7: e41081.
2. Bianconi, E., Piovesan, A., Facchin, F., Beraudi, A., Casadei, R., Frabetti, F., Vitale, L., Pelleri, M. C., Tassani, S., Piva, F., Perez-Amodio, S., Strippoli, P. and Canaider, S. (2013). An estimation of the number of cells in the human body. Ann Hum Biol 40(6): 463-471.
3. Cubi, R., Vembar, S. S., Biton, A., Franetich, J. F., Bordessoulles, M., Sossau, D., Zanghi, G., Bosson-Vanga, H., Benard, M., Moreno, A., Dereuddre-Bosquet, N., Le Grand, R., Scherf, A. and Mazier, D. (2017). Laser capture microdissection enables transcriptomic analysis of dividing and quiescent liver stages of Plasmodium relapsing species. Cell Microbiol 19(8): e12735.
4. Dembele, L., Gego, A., Zeeman, A. M., Franetich, J. F., Silvie, O., Rametti, A., Le Grand, R., Dereuddre-Bosquet, N., Sauerwein, R., van Gemert, G. J., Vaillant, J. C., Thomas, A. W., Snounou, G., Kocken, C. H. and Mazier, D. (2011). Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery. PLoS One 6(3): e18162.
5. Gupta, D. K., Dembele, L., Voorberg-van der Wel, A., Roma, G., Yip, A., Chuenchob, V., Kangwanrangsan, N., Ishino, T., Vaughan, A. M., Kappe, S. H., Flannery, E. L., Sattabongkot, J., Mikolajczak, S., Bifani, P., Kocken, C. H. and Diagana, T. T. (2019). The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development. Elife 8: e43362.
6. Gural, N., Mancio-Silva, L., Miller, A. B., Galstian, A., Butty, V. L., Levine, S. S., Patrapuvich, R., Desai, S. P., Mikolajczak, S. A., Kappe, S. H. I., Fleming, H. E., March, S., Sattabongkot, J. and Bhatia, S. N. (2018). In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium vivax Hypnozoites. Cell Host Microbe 23(3): 395-406 e394.
7. Joyner, C., Moreno, A., Meyer, E. V., Cabrera-Mora, M., Ma, H. C., Kissinger, J. C., Barnwell, J. W. and Galinski, M. R. (2016). Plasmodium cynomolgi infections in rhesus macaques display clinical and parasitological features pertinent to modelling vivax malaria pathology and relapse infections.Malar J 15(1): 451.
8. Kocken, C. H., van der Wel, A. and Thomas, A. W. (1999). Plasmodium cynomolgi: transfection of blood-stage parasites using heterologous DNA constructs. Exp Parasitol 93(1): 58-60.
9. Krotoski, W. A., Garnham, P. C., Bray, R. S., Krotoski, D. M., Killick-Kendrick, R., Draper, C. C., Targett, G. A. and Guy, M. W. (1982). Observations on early and late post-sporozoite tissue stages in primate malaria. I. Discovery of a new latent form of Plasmodium cynomolgi (the hypnozoite), and failure to detect hepatic forms within the first 24 hours after infection. Am J Trop Med Hyg 31(1): 24-35.
10. Natarajan, R., Thathy, V., Mota, M. M., Hafalla, J. C., Menard, R. and Vernick, K. D. (2001). Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasites. Cell Microbiol 3(6): 371-379.
11. Pasini, E. M., Bohme, U., Rutledge, G. G., Voorberg-Van der Wel, A., Sanders, M., Berriman, M., Kocken, C. H. and Otto, T. D. (2017). An improved Plasmodium cynomolgi genome assembly reveals an unexpected methyltransferase gene expansion. Wellcome Open Res 2: 42.
12. Posfai, D., Sylvester, K., Reddy, A., Ganley, J. G., Wirth, J., Cullen, Q. E., Dave, T., Kato, N., Dave, S. S. and Derbyshire, E. R. (2018). Plasmodium parasite exploits host aquaporin-3 during liver stage malaria infection. PLoS Pathog 14(5): e1007057.
13. Prudencio, M., Rodrigues, C. D., Ataide, R. and Mota, M. M. (2008). Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry. Cell Microbiol 10(1): 218-224.
14. Roth, A., Maher, S. P., Conway, A. J., Ubalee, R., Chaumeau, V., Andolina, C., Kaba, S. A., Vantaux, A., Bakowski, M. A., Thomson-Luque, R., Adapa, S. R., Singh, N., Barnes, S. J., Cooper, C. A., Rouillier, M., McNamara, C. W., Mikolajczak, S. A., Sather, N., Witkowski, B., Campo, B., Kappe, S. H. I., Lanar, D. E., Nosten, F., Davidson, S., Jiang, R. H. Y., Kyle, D. E. and Adams, J. H. (2018). A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum. Nat Commun 9(1): 1837.
15. Sattabongkot, J., Yimamnuaychoke, N., Leelaudomlipi, S., Rasameesoraj, M., Jenwithisuk, R., Coleman, R. E., Udomsangpetch, R., Cui, L. and Brewer, T. G. (2006). Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax. Am J Trop Med Hyg 74(5): 708-715.
16. Schmidt, L. H. (1983). Appraisals of compounds of diverse chemical classes for capacities to cure infections with sporozoites of Plasmodium cynomolgi. Am J Trop Med Hyg 32(2): 231-257.
17. Tarun, A. S., Baer, K., Dumpit, R. F., Gray, S., Lejarcegui, N., Frevert, U. and Kappe, S. H. (2006). Quantitative isolation and in vivo imaging of malaria parasite liver stages. Int J Parasitol 36(12): 1283-1293.
18. Voorberg-van der Wel, A. M., Zeeman, A. M., Nieuwenhuis, I. G., van der Werff, N. M., Klooster, E. J., Klop, O., Vermaat, L. C., Kumar Gupta, D., Dembele, L., Diagana, T. T. and Kocken, C. H. M. (2020). A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation. Commun Biol 3: 7.
19. Voorberg-van der Wel, A., Roma, G., Gupta, D. K., Schuierer, S., Nigsch, F., Carbone, W., Zeeman, A. M., Lee, B. H., Hofman, S. O., Faber, B. W., Knehr, J., Pasini, E., Kinzel, B., Bifani, P., Bonamy, G. M. C., Bouwmeester, T., Kocken, C. H. M. and Diagana, T. T. (2017). A comparative transcriptomic analysis of replicating and dormant liver stages of the relapsing malaria parasite Plasmodium cynomolgi.Elife 6: e29605.
20. Voorberg-van der Wel, A., Zeeman, A. M., van Amsterdam, S. M., van den Berg, A., Klooster, E. J., Iwanaga, S., Janse, C. J., van Gemert, G. J., Sauerwein, R., Beenhakker, N., Koopman, G., Thomas, A. W. and Kocken, C. H. (2013). Transgenic fluorescent Plasmodium cynomolgi liver stages enable live imaging and purification of Malaria hypnozoite-forms. PLoS One 8(1): e54888.
21. Yan, G., Zhang, G., Fang, X., Zhang, Y., Li, C., Ling, F., Cooper, D. N., Li, Q., Li, Y., van Gool, A. J., Du, H., Chen, J., Chen, R., Zhang, P., Huang, Z., Thompson, J. R., Meng, Y., Bai, Y., Wang, J., Zhuo, M., Wang, T., Huang, Y., Wei, L., Li, J., Wang, Z., Hu, H., Yang, P., Le, L., Stenson, P. D., Li, B., Liu, X., Ball, E. V., An, N., Huang, Q., Zhang, Y., Fan, W., Zhang, X., Li, Y., Wang, W., Katze, M. G., Su, B., Nielsen, R., Yang, H., Wang, J., Wang, X. and Wang, J. (2011). Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques. Nat Biotechnol 29(11): 1019-1023.
22. Zeeman, A. M., Lakshminarayana, S. B., van der Werff, N., Klooster, E. J., Voorberg-van der Wel, A., Kondreddi, R. R., Bodenreider, C., Simon, O., Sauerwein, R., Yeung, B. K., Diagana, T. T. and Kocken, C. H. (2016). PI4 Kinase Is a Prophylactic but Not Radical Curative Target in Plasmodium vivax-Type Malaria Parasites. Antimicrob Agents Chemother 60(5): 2858-63.
23. Zeeman, A. M., van Amsterdam, S. M., McNamara, C. W., Voorberg-van der Wel, A., Klooster, E. J., van den Berg, A., Remarque, E. J., Plouffe, D. M., van Gemert, G. J., Luty, A., Sauerwein, R., Gagaring, K., Borboa, R., Chen, Z., Kuhen, K., Glynne, R. J., Chatterjee, A. K., Nagle, A., Roland, J., Winzeler, E. A., Leroy, D., Campo, B., Diagana, T. T., Yeung, B. K., Thomas, A. W. and Kocken, C. H. (2014). KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.Antimicrob Agents Chemother 58(3): 1586-95.