5. The manufacturers’ manual of the CellTiter-Glo recommends the use of 100 μ L of CellTiter-Glo reagents for the 96-well cell culture plate assay. However, we can decrease the amount of CellTiter-Glo reagents to 25 μ L/well without affecting the luminescence [11].
Acknowledgments
This study was financially supported by the Japan Society for the Promotion of Science (JSPS), Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and the AMED/JICA, SATREPS.
References
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(2005) Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote- harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice. Cell Microbiol 7:383–392
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high-throughput drug screening targeting the intracellular stage of Trypanosoma cruzi, the agent of Chagas’ disease. Antimicrob Agents Chemother 54:3326–3334
4. Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M (2004) Simple and inexpensive fluo- rescence-based technique for high- throughput antimalarial drug screening. Antimicrob Agents Chemother 48:1803–1806
5. Sykes ML, Baell JB, Kaiser M, Chatelain E, Moawad SR, Ganame D et al (2012)
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proliferative activity against Trypanosoma bru- cei brucei strain 427 by a whole cell viability based HTS campaign. PLoS Negl Trop Dis 6:e1896
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7. Sykes ML, Avery VM (2009) A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screen- ing of Trypanosoma brucei brucei bloodstream form strain 427. Parasit Vectors 2:54
8. Sykes ML, Avery VM (2009) Development of an Alamar Blue viability assay in 384-well for- mat for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427. Am J Trop Med Hyg 81:665–674
9. Van Reet N, Pyana P, Rogé S, Claes F, Büscher P (2013) Luminescent multiplex viability assay
for Trypanosoma brucei gambiense. Parasit Vectors 6:207
10. Gillingwater K, Büscher P, Brun R (2007) Establishment of a panel of reference Trypanosoma evansi and Trypanosoma equiper- dum strains for drug screening. Vet Parasitol 148:114–121
11. Suganuma K, Allamanda P, Hakimi H, Zhou M, Angeles JM, Kawazu S et al (2014) Establishment of ATP-based luciferase viability assay in 96-well plate for Trypanosoma congo- lense. J Vet Med Sci 76:1437–1441
12. Hirumi H, Hirumi K (1991) In vitro cultiva- tion of Trypanosoma congolense bloodstream forms in the absence of feeder cell layers.
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13. Suganuma K, Sarwono AE, Mitsuhashi S, Jąkalski M, Okada T, Nthatisi M et al (2016) Mycophenolic acid and its derivatives as poten- tial chemotherapeutic agents targeting inosine monophosphate dehydrogenase in Trypanosoma congolense. Antimicrob Agents Chemother 60(7):4391–4393
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Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_9, © Springer Science+Business Media LLC 2017
Chapter 9
SYBR® Green I-Based Fluorescence Assay to Assess Cell Viability of Malaria Parasites for Routine Use in Compound Screening
Maria Leidenberger, Cornelia Voigtlọnder, Nina Simon, and Barbara Kappes
Abstract
Owing to its fast and reliable assessment of parasite growth, the SYBR® Green I-based fluorescence assay is widely used to monitor drug susceptibility of malaria parasites. Its particular advantages are that it is a simple, one-step procedure and very cost-effective making it especially suited for high through put screen- ing of newly developed drugs and drug combinations. Here we describe a SYBR® Green I-based fluores- cence assay protocol to be used for routine screening of compounds and extracts in a research laboratory environment. A variation of the standard protocol is also provided allowing to address stage-specific effects of fast-acting drugs.
Key words Fluorescence assay, SYBR Green, Growth inhibition assay, Malaria, Plasmodium falciparum, Compound screening
1 Introduction
All in vitro antimalarial drug sensitivity assays are based on the in vitro culture technique developed by Trager and Jensen (1976) allowing for a continuous culture of Plasmodium falciparum in a blood medium-mixture under controlled atmospheric conditions (see Subheading 3.3) [1]. This continuous culture technique enabled the development of several antimalarial drug sensitivity assay systems that are inter alia used (a) to screen for novel or improved chemical entities with antimalarial activity, (b) to examine the interaction of drug combinations, (c) to monitor the emergence of drug-resistant parasite strains, and (d) to determine the baseline sensitivity to new drugs before their market launch in a country.
The [3H]hypoxanthine incorporation assay developed in 1979 by Desjardins and coworkers has been the gold standard for antimalarial drug screening over decades [2]. Numerous
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antimalarial drug sensitivity microtiter plate assays have been developed since then omitting the use of radioactivity for the detection of parasite growth inhibition [3–10]. A detailed description of a number of these assays is provided in a compre- hensive book by Basco [11]. Therefore only two of these assays, which are widely used in the community, will be mentioned here. In 1993, Makler et al. established a novel colorimetric screening technique based on the activity of the parasite lactate dehydrogenase (pLDH) and the commercially available Malstat™ reagent [12]. In brief, 3- acetylpyridine adenine dinu- cleotide (APAD), a nicotinamide adenine dinucleotide (NAD) analogon, is reduced to APADH by pLDH, which in a subse- quent reaction is used to reduce the yellow tetrazolium salt, nitroblue tetrazolium, to the blue formazan product in the presence of phenazine ethosulfate. Reaction progress is detected by measuring the absorbance at 650 nm in a plate reader. In 2002, Noedl and coworkers adapted a commercially available enzyme-linked immunosorbent assay (ELISA) based on mono- clonal antibodies directed against P. falciparum histidine-rich protein II to malaria drug sensitivity testing [7]. Although widely used both assays are, however, not advisable for high- throughput screening (HTS), since they are too expensive or involve multistep procedure [13, 14]. This problem was resolved with the introduction of the SYBR green I dye-based fluores- cence assay that has been developed by Smilkstein and cowork- ers in 2004 [9]. This assay is suited for HTS and has been reported to be as sensitive as the [3H]hypoxanthine incorpora- tion assay [13]. Since its implementation, the SYBR green I fluorescence assay has been extensively validated and compared with existing methods and is nowadays for most applications the method of choice among the in vitro assays for testing the sen- sitivity of the human malaria parasites to drugs [13–17].
The SYBR green I fluorescence assay relies on the binding of SYBR green I to parasite DNA. It preferentially binds to double- stranded versus single-stranded DNA or RNA [18]. The fluorescence intensity therefore reflects the amount of double- stranded DNA in an individual sample. Mature erythrocytes are acaryotes lacking a nucleus and accordingly DNA and RNA. Thus the fluorescence signal detected in parasite cultures originates from the binding of the dye to parasite DNA in any of the eryth- rocytic stages of the parasite [4]. The fluorescence intensity can be determined using a minifluorometer, a fluorescence spectro- photometer, or a HTS versatile fluorescence-activated micro- plate reader.
Maria Leidenberger et al.
2 Materials
Prepare all solutions with ultrapure water from a Milli-Q water filtration station (sensitivity of 18 M Ω at 25 °C) and analytical grade reagents. Unless otherwise indicated reagents are prepared and kept at room temperature (RT). Cell culture medium and solutions for the treatment of parasite cultures have to be sterile.