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Jan 2021
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Plasmodium cynomolgi Berok Growth Inhibition Assay by Thiol-reactive Probe Based Flow Cytometric Measurement
硫醇反应探针流式细胞术检测食蟹猴疟原虫生长抑制   

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Abstract

The relapsing malaria species, Plasmodium vivax, is the most widely distributed and difficult-to-treat cause of human malaria. The merozoites of P. vivax preferentially invade ephemeral human CD71+ reticulocytes (nascent reticulocytes), thereby limiting the development of a robust continuous culture in vitro. Fortunately, P. vivax’s sister species, P. cynomolgi Berok, can be cultured continuously, providing the ability to screen novel therapeutics drug and vaccine candidates in a reliable and high-throughput manner. Based on well-established growth inhibition activity (GIA) assays against P. falciparum and P. knowlesi, this protocol adopts the current flow cytometry assay methodology and investigates P. vivax inhibitory antibodies using the P. cynomolgi Berok invasion model based on the thiol-reactivity and DNA abundance of viable parasites in macaque erythrocytes. Established GIA assays screen antibodies at either a single concentration or high/low dose concentrations to provide quick insights for prioritizing potential antibodies capable of specifically interrupting parasite ligand and host receptor binding with minimal concentrations. Hence, this protocol expands on the existing GIA assay by using serially diluted antibodies and generating a dose-response curve to better quantify the inhibitory efficacy amongst selected vaccine candidates.

Keywords: Plasmodium vivax (间日疟原虫), Plasmodium cynomolgi Berok (疟原虫), Malaria (疟疾), Growth inhibition activity assay (生长抑制活性测定), Antibody inhibition (抗体抑制), Invasion biology (入侵生物学)

Background

The growth inhibition assay examines the re-invasion and growth of the Plasmodium spp. over two cycles of parasite replication (one cycle is approximately 24 h for P. knowlesi and 48 h for P. vivax, P. cynomolgi, and P. falciparum). After egress, asexual merozoites are released to continuously invade and multiply within host erythrocytes, increasing parasitaemia. During merozoite invasion, a cascade of parasitic proteins interacts with host cell receptors to manipulate host cell entry. In asymptomatic P. vivax patients, monoclonal antibodies recognise a wide range of parasitic ligands such as the duffy binding proteins (DBPs), reticulocyte binding proteins (RBPs), merozoite surface proteins (MSPs), and apical membrane antigen (AMA) (Han et al., 2019; Rawlinson et al., 2019). These antibodies have been shown to be strain-transcendent, confer protective immunity, and reduce merozoite invasion to reticulocytes in vitro short-term culture (Russell et al., 2011). However, the lack of a successfully multiplying continuous culture system of P. vivax reduces the detection signal in the growth inhibition assay (GIA) (Gunalan et al., 2020). To overcome this limitation, past P. vivax studies utilized surrogate species such as P. knowlesi, P. cynomolgi, and P. falciparum to screen inhibitory antibodies (Mitran et al., 2019; Collins et al., 1999; Muh et al., 2020).


The conventional GIA quantifies parasite viability and density using the SYBR green dye and lactate dehydrogenase (LDH) enzyme via flow cytometry and spectrophotometry, respectively (Mohring et al., 2020). Our approach explores thiol reactivity quantification in P. cynomolgi, which reliably assesses P. falciparum parasitaemia in antibody-dependent cellular inhibition assays (Jogdand et al., 2012). The quantification is based on the accumulation and fluorescent covalent biconjugates formed by the thiol-reactive probe carbocyanin, which acts as an indicator for the parasite’s viability in the sarcoplasmic reticulum and mitochondria of eukaryotes (Habicht and Brune 1980; Amaratunga et al., 2014). As the Mitotracker product is available in a wide range of fluorescent dyes and probes, this method also presents an opportunity to investigate other fluorescent channels in flow cytometry. Furthermore, our approach adapts the mean inhibitory concentration approach commonly used in drug assays, allowing researchers to select amongst highly effective inhibitory antibodies with low inhibitory concentrations.

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 ScientificTM, 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, GlutaMAXTM 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.

References

  1. Amaratunga, C., Neal, A. T. and Fairhurst, R. M. (2014). Flow cytometry-based analysis of artemisinin-resistant Plasmodium falciparum in the ring-stage survival assay. Antimicrob Agents Chemother 58(8): 4938-4940.
  2. Chua, A. C. Y., Ong, J. J. Y., Malleret, B., Suwanarusk, R., Kosaisavee, V., Zeeman, A. M., Cooper, C. A., Tan, K. S. W., Zhang, R., Tan, B. H., Abas, S. N., Yip, A., Elliot, A., Joyner, C. J., Cho, J. S., Breyer, K., Baran, S., Lange, A., Maher, S. P., Nosten, F., Bodenreider, C., Yeung, B. K. S., Mazier, D., Galinski, M. R., Dereuddre-Bosquet, N., Le Grand, R., Kocken, C. H. M., Renia, L., Kyle, D. E., Diagana, T. T., Snounou, G., Russell, B. and Bifani, P. (2019). Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages. Nat Commun 10(1): 3635.
  3. Collins, W. E., Warren, M. and Galland, G. G. (1999). Studies on infections with the Berok strain of Plasmodium cynomolgi in monkeys and mosquitoes. J Parasitol 85(2): 268-272.
  4. Gunalan, K., Rowley, E. H. and Miller, L. H. (2020). A Way Forward for Culturing Plasmodium vivax. Trends Parasitol 36(6): 512-519.
  5. Habicht, J. and Brune, K. (1980). Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells. Exp Cell Res 125(2): 514-518.
  6. Han, J. H., Cheng, Y., Muh, F., Ahmed, M. A., Cho, J. S., Nyunt, M. H., Jeon, H. Y., Ha, K. S., Na, S., Park, W. S., Hong, S. H., Shin, H. J., Russell, B. and Han, E. T. (2019). Inhibition of parasite invasion by monoclonal antibody against epidermal growth factor-like domain of Plasmodium vivax merozoite surface protein 1 paralog. Sci Rep 9(1): 3906.
  7. Jogdand, P. S., Singh, S. K., Christiansen, M., Dziegiel, M. H., Singh, S. and Theisen, M. (2012). Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition. Malar J 11: 235.
  8. Moll, K., L. M. I., Perlmann, H., Scherf, A. and Wahlgren, M. (2008). Methods In Malaria Research. Malaria Research and Reference Reagent Resource Center (MR4) American Type Culture Collection (ATCC). 5th edition.
  9. Mitran, C. J., Mena, A., Gnidehou, S., Banman, S., Arango, E., Lima, B. A. S., Lugo, H., Ganesan, A., Salanti, A., Mbonye, A. K., Ntumngia, F., Barakat, K., Adams, J. H., Kano, F. S., Carvalho, L. H., Maestre, A. E., Good, M. F. and Yanow, S. K. (2019). Antibodies to Cryptic Epitopes in Distant Homologues Underpin a Mechanism of Heterologous Immunity between Plasmodium vivax PvDBP and Plasmodium falciparum VAR2CSA. mBio 10(5): ):e02343-19.
  10. Mohring, F., Rawlinson, T. A., Draper, S. J. and Moon, R. W. (2020). Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi. Bio-protocol 10(17): e3743.
  11. Muh, F., Kim, N., Nyunt, M. H., Firdaus, E. R., Han, J. H., Hoque, M. R., Lee, S. K., Park, J. H., Moon, R. W., Lau, Y. L., Kaneko, O. and Han, E. T. (2020). Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi. PLoS Negl Trop Dis 14(6): e0008323.
  12. Rawlinson, T. A., Barber, N. M., Mohring, F., Cho, J. S., Kosaisavee, V., Gerard, S. F., Alanine, D. G. W., Labbe, G. M., Elias, S. C., Silk, S. E., Quinkert, D., Jin, J., Marshall, J. M., Payne, R. O., Minassian, A. M., Russell, B., Renia, L., Nosten, F. H., Moon, R. W., Higgins, M. K. and Draper, S. J. (2019). Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody. Nat Microbiol 4(9): 1497-1507.
  13. Russell, B., Suwanarusk, R., Borlon, C., Costa, F. T., Chu, C. S., Rijken, M. J., Sriprawat, K., Warter, L., Koh, E. G., Malleret, B., Colin, Y., Bertrand, O., Adams, J. H., D'Alessandro, U., Snounou, G., Nosten, F. and Renia, L. (2011). A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax. Blood 118(13): e74-81.
  14. Shen, F. H., Ong, J. J. Y., Sun, Y. F., Lei, Y., Chu, R. L., Kassegne, K., Fu, H. T., Jin, C., Han, E. T., Russell, B., Han, J. H. and Cheng, Y. (2021). A chimeric Plasmodium vivax merozoite surface protein antibody recognizes and blocks erythrocytic P. cynomolgi berok merozoites in Vitro. Infect Immun 89(2): e00645-20.

简介

[摘要]在复发疟疾的物种,疟原虫间日疟原虫,是分布最广,不易-到-人类疟疾的治疗原因。间日疟原虫的裂殖子优先侵入短暂的人类 CD71 +网织红细胞(新生网织红细胞),从而限制了体外稳健连续培养的发展。幸运的是,间日疟原虫的妹妹种,P.食蟹猴 Berok 可以连续培养,从而能够以可靠和高通量的方式筛选新型治疗药物和候选疫苗。基于针对恶性疟原虫和诺氏疟原虫的成熟生长抑制活性 (GIA) 测定,该协议采用当前的流式细胞术测定方法,并使用基于硫醇的食蟹猴Berok 入侵模型研究间日疟原虫抑制抗体猕猴红细胞中活寄生虫的反应性和 DNA 丰度。建立GIA测定筛选在一个单一的浓度或高/低剂量浓度的抗体,以提供快速的见解为优先能够特异性阻断寄生虫配体和宿主受体具有最小浓度的结合潜在抗体小号。因此,该协议通过使用连续稀释的抗体并生成剂量反应曲线来扩展现有的 GIA 检测,以更好地量化所选候选疫苗的抑制功效。


[背景]的生长抑制分析检查的再次侵袭和生长疟原虫属。在寄生虫复制的两个周期(Ô NE周期为大约24小时诺氏疟原虫和48小时间日疟原虫,P.食蟹猴,和恶性疟原虫)。排出后,无性裂殖子被释放,在宿主红细胞内不断侵入和繁殖,增加寄生虫血症。在裂殖子入侵期间,一系列寄生蛋白与宿主细胞受体相互作用以操纵宿主细胞进入。在无症状间日疟患者中,单克隆抗体可识别多种寄生配体,例如达菲结合蛋白 (DBP)、网织红细胞结合蛋白 (RBP)、裂殖子表面蛋白 (MSP) 和顶膜抗原 (AMA) (Han等人,2019 年;罗林森等人,2019 年)。这些抗体已经被证明是应变超越,保护性免疫,并减少裂殖子侵入到网织红细胞体外短期-长期培养(罗素等人,2011) 。然而,缺乏间日疟原虫成功繁殖的连续培养系统降低了生长抑制试验 (GIA) 中的检测信号(Gunalan等,2020)。为了克服这一限制,过去的间日疟研究使用替代物种,如P. kno wlesi、P. cynomolgi和P. falciparum来筛选抑制性抗体(Mitran等人,2019 年;Collins等人,1999 年;Muh等人. , 2020) 。

常规 GIA分别通过流式细胞术和分光光度法使用 SYBR 绿色染料和乳酸脱氢酶 (LDH) 来量化寄生虫的生存力和密度(Mohring等人,2020 年)。我们的方法探讨了巯基反应性定量P.食蟹猴,这可靠地的Assesse小号恶性疟原虫抗体寄生虫血症-依赖性细胞抑制测定法(Jogdand等人,2012) 。量化基于由硫醇反应性探针碳蓝蛋白形成的积累和荧光共价双结合物,碳蓝蛋白作为寄生虫在真核生物的肌浆网和线粒体中的生存能力的指示剂(Habicht 和 Brune 1980;Amaratunga等人,2014) 。由于 Mitotracker 产品可用于多种荧光染料和探针,因此该方法也提供了研究流式细胞术中其他荧光通道的机会。此外,我们的方法采用了药物测定中常用的平均抑制浓度方法,使研究人员能够在低抑制浓度的高效抑制抗体中进行选择。

关键字:间日疟原虫, 疟原虫, 疟疾, 生长抑制活性测定, 抗体抑制, 入侵生物学

 
材料和试剂
 
1. 1.5 ml 微量离心管(Axygen ® ,目录号:MCT-150-C)      
2. 15 ml 离心管(Corning,Costar,目录号:CLS430052)      
3. 50 ml 离心管(Corning,Costar,目录号:CLS430291)      
4. 24 孔平底板(Corning,Costar,目录号:CLS3738)      
5. 96 孔圆底板(Corning,Costar,目录号:CLS3367)      
6. 96 孔平底板(Corning,Costar,目录号:CLS3370)      
7.非排气 T75 烧瓶(Corning,Costar,目录号:CLS3814)      
8. 3 ml 圆底聚苯乙烯管(Becton Dickinson ,目录号:156758)      
9.无菌 V 通道储液器(VWR,目录号:VWRI613-1174)      
10.无菌14毫米培养皿(VWR,目录号:VWRI390-1373)   
11.无纺布过滤器(Antoshin)   
12.铝箔   
13.锂肝素真空吸尘器(Becton Dickinson,目录号:367880)   
14. Clot activator vacuteiners(Becton Dickinson,目录号:367820)   
15. P. cynomolgi Berok 红细胞培养(Bruce Russell,奥塔哥大学)(Chua等人,2019)   
16. Macaca fascicularis erythrocytes (Monash Animal Research Platform, Monash University)   
17.米。束状血清(Monash Animal Research Platform, Monash University)   
18. Pre-immune 和P. vivax抗体 (Yang Cheng, 江南大学) (Shen et al. , 2021)   
19.补充有 GlutaMAX(Gibco,目录号:61870-036)的 RPMI 1640 培养基   
20. HEPES(Sigma-Aldrich,目录号:H4034)   
21. D-葡萄糖(Sigma-Aldrich,目录号:G7021)   
22.次黄嘌呤(Calbiochem,目录号:4010BC)   
23. PBS 片剂(Gibco,目录号:18912014 )   
24. Giemsa(VWR,目录号:352603R)   
25.甲醇(Fisher Scientific,目录号:A412-4)   
26. Hoechst(Sigma-Aldrich,目录号:63493)   
27. Mitotracker Deep Red(Thermo Fisher Scientific,FM Molecular Probes,目录号:22426)   
28.完整培养基(见食谱)   
 
设备
 
II类微生物安全柜
微孔板轨道振荡器
多通道移液器(12 通道移液器,10-100 μl)(Thermo Scientific TM ,目录号:4661070N)
台式平板离心机(Eppendorf,型号:5804R )
带 100 ×油浸镜头的光学显微镜(NIKON,型号:YS100)
Canto II 流式细胞仪(Becton Dickinson,Canto II)
将气体与 5% O 2 、5% CO 2和其余与 N 2 混合
缺氧培养箱(Stemcell,目录号:27310)
37 ℃培养箱
4 °C冰箱
-20 °C冰箱
 
软件
 
FACSDiva 6.1.3 软件(Becton Dickinson)
Flowjo, LLC (Becton Dickinson, Tree Star Inc, https://www.flowjo.com/contact )
Microsoft Excel(微软,https://www.microsoft.com/en-us/microsoft-365/excel)
Prism 8(GraphPad,https ://www.graphpad.com/scientific-software/prism/ )
 
程序
 
P arasite文化
解冻一含有冷冻小瓶1 ml的的P.食蟹猴Berok寄生使用一系列以逐滴方式氯化钠溶液红细胞,根据莫尔等人。( 2008) 。
在 24 孔板中的完整培养基中重新悬浮颗粒状寄生红细胞,并在 37 °C培养箱中充满三混合气体的缺氧培养箱中以 5% 血细胞比容培养寄生虫培养物。
过夜孵育后,在有25厘米扩大在非排气式烧瓶中的寄生的红细胞之前检查成功的寄生虫成熟2使用在5%血细胞比容的细胞培养烧瓶中号。食蟹猴红细胞,根据蔡氏等人。( 2019) 。
如果寄生虫培养显示š早期滋养体和裂殖体阶段的混合物(1:1)寄生的红细胞中的实验天前2天,执行一个“快速和肮脏的”培养的同步。
 
“又快又脏”的文化同步
实验前约 30 小时,在室温下以 1,114 × g沉淀寄生虫培养物(至少 2 ml 培养物,5% 血细胞比容)2 分钟。
将上清液(完全培养基)置于单独的 50 ml Falcon 管中。
小心吸并除去沉淀的红细胞(裂殖的棕黑色着色顶层即用移液管手动没有egressed)。
使用新的移液器吸头,用先前留出的上清液轻轻地重悬沉淀的红细胞,然后将混合物转移到无排气孔的 T75 烧瓶中。气体与三混合气体 5 秒,并在 37 °C下孵育烧瓶过夜。
检查隔夜寄生的红细胞是否在第二天下午成熟到裂殖体阶段(至少 70%)。
 
GIA 抗体制备
在冰上解冻冷冻的抗体小瓶,并在寄生红细胞培养物中确认裂殖体成熟后准备新鲜的。
在抗体稀释板中,用 200 μl 无菌水填充 96 孔圆底板的所有外孔。
注意:如果使用平孔底板,请将板放置在一定角度,以确保在每个孔的底部进行一致的移液。
标记板indicat的盖Ë与抗体名称的行和列从“1”到“10”。
用 20 μl 完整培养基标记“2”到“10”的种子孔(参见图 1A 的板布局)。
用培养基将相应的抗体稀释到各自标有“1”的孔中,最终体积为 30 μl,浓度为1 mg/ml。
通过将 10 μl 从“1”孔转移到“2”孔进行三倍连续稀释。通过移液混合。
重复抗体的连续稀释,直至孔“10” 。
 
 
图 1. 抗体和实验板布局。蓝色结肠ü红井显示装有200微升无菌水打井。空白表示为“X”。A.带有标签的抗体板布局图,从 A 行到 H 行表示抗体。第 1 到 12 列表示抗体浓度下降。橙色的colo ü红井表示20个微升完全培养基(CM)作为抗稀释剂。B.实验板布局的插图,带有标签指示来自 A 行到 H 行的抗体和浓度从 1 到 10。B 行包含阳性对照,G 行包含阴性对照。深红色孔表示含有感染的孔红细胞 (iRBC) ,浅红色孔表示未感染的红细胞 (uRBC)。
 
P.食蟹Berok GIA -设置了
在设置之前,确保大多数寄生红细胞处于中晚期裂殖体阶段(培养中至少 70% 的寄生红细胞,每个裂殖体至少含有8 个裂殖子)。
为了尽量减少培养基蒸发,用 200 μl 无菌水填充 96 孔圆底或平底板的所有外孔,并在缺氧培养箱中放置一个装有无菌水的14 mm培养皿。
用“1”至“10”孔标记板盖,以一式两份标明抗体名称及其相应的测试稀释度。包括一排阳性和阴性对照孔(参见图 1B 的板布局)。
种子阴性对照孔与 70 μl未感染的红细胞在 1% 血细胞比容。
在 1% 血细胞比容和 0.5%寄生虫血症下接种70 μl寄生红细胞的种子阳性对照孔。
每个测试孔的最终体积为 70 μl。Ç alculate沉淀的红细胞的体积为70的最终体积微升/孔在1%血细胞比容和0.5%的寄生虫血症。然而,由于抗体将被稀释 10倍以达到 100 μg/ml 的最终浓度,因此用 63 μl/孔的完整培养基体积重新悬浮沉淀的红细胞。
将混合物转移到无菌V 型槽中。
在将63 μl 寄生虫培养物移入每个测试孔之前,通过轻轻上下移液来混合储液器中的溶液,以确保溶液均匀。
使用多通道移液器将 7 μl 抗体(来自步骤 C6)添加到每个孔中,一式两份。
在37 °C 下用三混合气体冲洗缺氧孵化器室中的板。
为了在此 2 周期测定中的裂殖子入侵的第二周期保持健康的寄生红细胞培养物,在孵育后 48 小时向每个孔中添加7 μl 完整培养基(含新鲜补充的血清)。
 
P. cynomolgi Berok GIA–染色
孵育后96 小时,轻轻重悬寄生虫培养物,然后将 20 μl 提取到新的 96 孔圆底板中进行染色。
通过在室温下以400 × g的速度在离心机中旋转平板5 分钟,以低减速使红细胞沉淀。
用 8 μM Hoechst 34580 和 150 nM Mitotracker Deep Red FM 在 1 × PBS 中制备最终体积为 5 0 μl/孔的染色 MasterMix 。
旋转完成后,从每个孔中取出尽可能多的上清液。
用 50 μl 染色 MasterMix 重悬沉淀的红细胞。
在室温下以 120 rpm 的速度在微孔板轨道振荡器上孵育板20 分钟。为防止染料淬灭,请用一大片铝箔(可重复使用)盖住摇床。
通过在室温下以400 × g的速度在离心机中旋转平板5 分钟,以低减速使红细胞沉淀。
去除尽可能多的上清液,并用 200 μl PBS洗涤细胞。
重复离心和洗涤步骤Ş两次。
在 200 μl PBS 中重悬细胞,并将混合物转移到 3 ml 圆底聚苯乙烯管中,以便在流式细胞仪上进行分析。
 
通过流式细胞术分析 Hoechst 34580 + /Mitotracker Deep Red FM +寄生红细胞
使用 FACS 测量寄生虫血症。
每个样本记录 50,000 到 100,000 个细胞。
将数据导出为 fsc 文件格式。
 
数据分析
 
使用Flowjo 软件,对单个细胞进行门控(x 轴:SSC-H,y 轴:FSC-H),然后根据单细胞群体选择相应的检测器通道并门控双阳性 Hoechst 34580 + /Mitotracker Deep Red +人口。
将门控人口值导出到 Excel 软件。
从测试样品中减去基线值(阴性对照)。
将值从 Excel 软件复制到 GraphPad Prism 8 软件。
为了比较不同药物浓度下 Hoechst 34580 + /Mitotracker Deep Red +活寄生虫的百分比,首先对药物浓度进行 log 2转换,然后将数据值标准化。
使用四参数逻辑模型或 log(抑制剂)与响应 - 可变斜率函数进行剂量响应曲线拟合。
使用单向方差分析和 Tukey 事后检验函数比较各种抗体的平均 IC 50值。
 
食谱
 
完整的文化媒体
RPMI-1640 培养基、GlutaMAX TM补充剂、HEPES 以及以下添加剂:
2.5 毫升 HEPES (1 M)
1.0 g/L D-(+)-葡萄糖
0.05 克/升次黄嘌呤
20% (vol/vol) M . 束状血清
无菌过滤器并储存在 4 °C
 
致谢
 
这窝K值是由医学科学的江原大学2020年研究所(JH H)的研究基金的支持,并通过从BR实验室(BR)的马斯登基金17 U00-241。该协议改编自沉等人。(2021)。
 
利益争夺
 
作者声明没有相互竞争的经济利益。
 
参考
 
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引用:Ong, J. J. Y., Russell, B. and Han, J. (2021). Plasmodium cynomolgi Berok Growth Inhibition Assay by Thiol-reactive Probe Based Flow Cytometric Measurement. Bio-protocol 11(17): e4147. DOI: 10.21769/BioProtoc.4147.
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