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Plasmodium falciparum Rosette Disruption Assay
恶性疟原虫莲座破坏试验   

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Abstract

Rosetting, i.e. the capacity of Plasmodium falciparum-infected red blood cells (iRBCs) to bind two or more uninfected red blood cells (RBCs) is associated with severe malaria in African children. Disruption of rosettes using small soluble inhibitors or specific antibodies is viewed as an interesting strategy to treat or prevent severe malaria manifestations. The protocol presented here describes an assay to monitor rosette dissociation, validated for the Palo Alto VarO, IT4/R29 and 3D7/PF13 rosetting clones (Vigan-Womas  et al., 2011).

Materials and Reagents

  1. Rosette-forming iRBCs
    1. Culture of rosette-forming parasites at mature stages (trophozoite to early schizont stages, 5-10% parasitaemia, 5% haematocrit)
    2. Parasite clonal lines 3D7/PF13, Palo Alto 89F5 VarO, IT4/R29 or patient isolates

  2. Culture medium reagents
    1. Malaria non-immune human AB+ serum (pool of 3-5 different donors)
    2. RPMI 1640 medium with L-glutamine and 25 mM HEPES (500 ml) (Life Technologies, Gibco®, catalog number: 52400 )
    3. 10 mM Hypoxanthine solution (100x) (C.C.Pro, catalog number: Z-41-M )
    4. Gentamicin solution (50 mg/ml) (Sigma-Aldrich, catalog number: G1397 )

  3. Other materials
    1. Non pyrogenic sterile polystyrene, Rectangular Canted Neck Cell Culture Flask with Plug Seal Cap (75 cm2, Corning Incorporated, catalog number: 430720 ; 25 cm2,  Corning Incorporated, catalog number: 430168 , for 20 and 5 ml culture medium, respectively)
    2. Ice cold Ficoll (LymphoprepTM, density 1.077 g/ml) (Abcys, catalog number: 1114545 )
    3. Dextran sulfate sodium salt from Leuconostoc spp, MW > 500,000 (Sigma-Aldrich, catalog number: D6001 )
    4. Heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3393 )
    5. Fucoidan from Fucus vesiculosus (Sigma-Aldrich, catalog number: F5631 )
    6. Hoechst 33342, 10 mg/ml solution in water (Life Technologies, Molecular Probes®, catalog number: H-3570 )
    7. Sterile distilled water RNase DNase free (Life Technologies, Gibco®, catalog number: 10977-035 )
    8. Mouse, rat or rabbit immune and pre-immune polyclonal sera
    9. Monoclonal antibodies (mAbs) reacting with the surface of rosette-forming iRBCs
    10. Serum or plasma samples from individuals living in a malaria endemic region
    11. Serum or plasma samples from malaria non-immune individuals
      Note that all plasma samples must be collected using an anticoagulant other than heparin (heparin disrupts rosettes of multiple strains at the concentration used for anticoagulant activity. Convenient anticoagulants are EDTA [ethylene diamine tetraacetic acid] or sodium citrate). Store at -20 °C or -80 °C until use.
      Note: Abs are home made or collected from patients.

Equipment

  1. 5 ml polystyrene round-bottom sterile tube (BD Biosciences, Falcon®, catalog number: 352058 )
  2. Sterile laboratory vacuum filter 0.22 µm (Stericup® Filter Units Millipore, catalog number:  051446 )
  3. Sterile polypropylene conical centrifuge tubes (15 ml, BD Biosciences, Falcon®, catalog number: 352097 ;  50 ml, BD Biosciences, Falcon®, catalog number: 352098 )
  4. Sterile disposable serological 1 ml aspiration pipette (Dominique Dutscher, catalog number:  999079 )
  5. Professional gloves (e.g. Kimtech Sterling Nitrile gloves, Kimberley-Clark Professional, catalog number: 99211 , or Satin Plus gloves Kimberley-Clark Professional, catalog number: SP2330E or 2220E )
  6. 1.4 ml matrix round bottom non sterile tubes (Thermo Fisher Scientific, catalog number:  10630784 )
  7. Microscope glass slides (Thermo Fisher Scientific, catalog number: 10090431 ) and cover slips 22 x 22 mm (Thermo Fisher Scientific, catalog number: 11728691 )
  8. Centrifuge with a swing bucket rotor (Thermo Fisher Scientific Heraeus Multifuge 3SR+ centrifuge )
  9. Centrifuge Eppendorf 5702 with an A-4-38 swing bucket rotor (Thermo Fisher Scientific, catalog number: 05-400-318)
  10. Vacuum pump (ILMVAC Biovac, model: 104 )
  11. Fluorescence microscope with UV-light and 40x or 100x magnification (Leica fluorescence microscope DM5000B, HP Plan ocular 10 x 22 507897, HCX Plan 100x Oil, HCXPL 40x PH2)
  12. Incubator at 37 °C in continuous gazing: 5% O2, 5% CO2 and 90% N2 (Thermo Fisher Scientific Binder incubator, model: CB210 )
  13. Laminar flow class II, type A2 biological safety cabinet (e.g. Thermo Fisher Scientific HeraSafe, catalog number: 13-998-002 )

Procedure

Please note that gloves must be used throughout the handling procedures.

  1. Preparation of rosettes
    1. Prepare parasite cultures of rosette-forming parasites (see "Plasmodium falciparum Rosette formation assay", sections 1 and 2). Prepare enough material to perform each assay in triplicate: Each assay requires 3 x 40 μl rosette-rich suspension at 2% haematocrit (see 1.i-j.). This corresponds to an initial 20 μl culture at 6-8% parasitemia i.e. approx.
    2. Harvest the cultures of rosette-forming parasites at mature trophozoite to schizont stages (5- 10% parasitaemia). Avoid late or segmented schizonts, which form loose rosettes.
    3. Enrich for rosettes by centrifugation on ice-cold Ficoll (see "Plasmodium falciparum Rosette formation assay"- section 2). Rosette disruption assays are best carried out with parasite cultures with more than 50% rosette frequency.
    4. Spin down the cells by centrifugation in the Fisher Scientific Heraeus Multifuge for 10 min at 256 x g (1,200 rpm), room temperature.
    5. Discard the supernatant by aspiration using a 1 ml aspiration sterile pipette connected to a vacuum pump.
    6. Gently resuspend the rosette-rich pellet (some rosettes tend to be disrupted by simple mechanical handling, avoid back and forth harsh pipetting) in 4 ml pre-warmed CCM (see Recipes) supplemented with 10 μg/ml Hoechst.
    7. Mix gently and incubate for 10 min at 37 °C in the dark in an incubator with continuous gazing (5% O2, 5% CO2, 90% N2).
    8. Wash once with CCM: Add 900 μl CCM, centrifuge 3 min at 256 x g (1,200 rpm), in the Fisher Scientific Heraeus Multifuge at room temperature.
    9. Remove the supernatant and resuspend the pellet at 2% haematocrit in CCM (2 volumes (vol.) cell pellet + 98 vol. CCM). Parasitaemia of the preparation is usually around 5%.
    10. Dispense 40 μl aliquots of the rosette-rich RBC suspension in 1.4 matrix round bottom tubes.
    11. Centrifuge, in the Fisher Scientific Heraeus Multifuge 5 min at 178 x g (1,000 rpm) room temperature.
    12. Discard the supernatant and process the pellet as described in section 2 or 3.
    13. Prepare serial dilutions (0.1 μg/ml - 1 mg/ml) of heparin, dextran sulfate, fucoidan or other sulphated glycans in CCM.
    14. Prepare serial dilutions of sera, plasma (starting dilution 1/5) or purified mAbs or immunoglobulin preparations in CCM as diluent. Include negative controls such as CCM alone and non-immune serum (or Ig) and a positive control with sera or mAbs able to disrupt rosettes.
  2. Dissociation assay with sulphated glycans
    1. Add 100 μl diluted sulphated glycan to the rosette preparation (section 1-j).
    2. Incubate for 30 min at 37 °C (use an incubator rather than water-bath).
    3. Remove 60 μl of medium and resuspend the sedimented pellet in the remaining volume.
    4. Take a 10 μl aliquot, put it onto a microscope slide and add a cover slip.
    5. Count under the microscope 100-200 iRBCs and score iRBCs engaged in rosettes (i.e. trophozoite or schizont stages having bound two or more uninfected RBCs) (Figure 1).

    Figure 1. Rosette dissociation assay before A and after B incubation with 10 μg/ml dextran sulphate.

    1. Calculate the rosetting rate = (No. mature stage iRBCs engaged in rosettes/No. mature stages) x 100.
    2. Calculate the dissociation rate = (rosetting rate of control culture without inhibitor - rosetting rate of treated sample/rosetting rate of control culture without inhibitor) x 100.
  3. Dissociation assay with immune reagents
    1. Prepare serial dilutions of sera, plasma (starting dilution 1/5) or purified mAbs or immunoglobulin preparations to be tested. Include negative controls such as CCM alone and non-immune serum (or Ig) and a positive control with sera or mAbs able to disrupt rosettes.
    2. Add 40 μl diluted serum or plasma to the rosette preparation (section 1-n).
    3. Incubate for 30 min at 37 °C (use an incubator rather than water-bath).
    4. Resuspend the sedimented pellet in the incubation medium.
    5. Take a 10 μl aliquot, put it onto a microscope slide and add a cover slip.
    6. Count under the microscope, 100-200 iRBCs and score iRBCs engaged in rosettes.
    7. Calculate the rosetting rate = (No. mature stage iRBCs engaged in rosettes/No. mature stages) x 100.
    8. Calculate the dissociation rate = (rosetting rate of control culture incubated with non immune reagent - rosetting rate of treated sample/rosetting rate of control culture incubated with non immune reagent) x 100.
      Note that at certain dilutions, polyclonal immune reagents (and IgM mAbs) tend to disrupt rosettes and agglutinate iRBCs. Immune agglutinates are easily visualised as densely packed iRBCs (see Figure 2). Dissociation is readily visualised at higher dilutions.


Figure 2. Agglutination of rosette-forming iRBCs obtained after incubation with mouse polyclonal antibodies directed against a PfEMP1-derived surface exposed domain (dilution 1/40). The iRBC nuclei were stained with Hoechst 33342 and cells were examined by light A and UV B microscopy.

Recipes

  1. Complete culture medium (CCM, 500 ml)
    445 ml RPMI 1640
    5 ml Hypoxanthine solution (final concentration, 100 μM)
    200 μl Gentamicin solution (final concentration 20 μg/ml)
    50 ml Human AB+ serum (final concentration 10%)
    Sterilize using a 0.22 μm filter unit
    Store at 4 °C or -20 °C for long-term storage
  2. Stock solutions of sulphated glycans
    Prepare a 10 mg/ml solution of sulphated glycan in sterile water (0.1 g + 10 ml water)
    Sterilize using a 0.22 μm filter unit
    Aliquot and store at -80 °C
    Use each aliquot once (do not freeze-thaw)

Acknowledgments

This protocol was adapted from the following publications: Vigan-Womas et al. (2008 and 2010). This work was supported by the Agence Nationale de la Recherche, contract ANR-07-MIME-021-0 (www.agence-nationale-recherche.fr/), and the 7th European Framework Program, FP7/2007-2013, (http://cordis.europa.eu/fp7/home_en.html) contract 242095, Evimalar.

References

  1. Vigan-Womas, I., Guillotte, M., Le Scanf, C., Igonet, S., Petres, S., Juillerat, A., Badaut, C., Nato, F., Schneider, A., Lavergne, A., Contamin, H., Tall, A., Baril, L., Bentley, G. A. and Mercereau-Puijalon, O. (2008). An in vivo and in vitro model of Plasmodium falciparum rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype. Infect Immun 76(12): 5565-5580.
  2. Vigan-Womas, I., Lokossou, A., Guillotte, M., Juillerat, A., Bentley, G., Garcia, A., Mercereau-Puijalon, O. and Migot-Nabias, F. (2010). The humoral response to Plasmodium falciparum VarO rosetting variant and its association with protection against malaria in Beninese children. Malar J 9: 267.
  3. Vigan-Womas, I., Guillotte, M., Juillerat, A., Vallieres, C., Lewit-Bentley, A., Tall, A., Baril, L., Bentley, G. A. and Mercereau-Puijalon, O. (2011). Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes. PLoS One 6(1): e16544.

简介

Rosetting,即恶性疟原虫感染的红细胞(iRBC)结合两种或更多未感染的红细胞(RBC)的能力与非洲儿童中的严重疟疾有关 。 使用小的可溶性抑制剂或特异性抗体破坏玫瑰花结被视为治疗或预防严重疟疾表现的有趣策略。 本文提供的方案描述了监测玫瑰花结解离的测定,验证用于Palo Alto VarO,IT4/R29和3D7/PF13玫瑰花结的克隆(Vigan-Womas等人,2011)。

材料和试剂

  1. 形成玫瑰花结的iRBC
    1. 在成熟阶段(滋养体至早期裂殖期,5-10%寄生虫血症,5%血细胞比容)的玫瑰花结形成寄生虫的培养物
    2. 寄生物克隆系3D7/PF13,Palo Alto 89F5 Var0,IT4/R29或患者分离株

  2. 培养基试剂
    1. 疟疾非免疫人AB血清+血清(3-5个不同供体的汇集物)
    2. 具有L-谷氨酰胺和25mM HEPES(500ml)的RPMI 1640培养基(Life Technologies,Gibco ,目录号:52400)
    3. 10mM次黄嘌呤溶液(100x)(C.C.Pro,目录号:Z-41-M)
    4. 庆大霉素溶液(50mg/ml)(Sigma-Aldrich,目录号:G1397)
  3. 其他材料
    1. 非致热无菌聚苯乙烯,具有塞密封盖的长方形颈部细胞培养瓶(75cm 2,Corning Incorporated,目录号:430720; 25cm 2), Corning Incorporated,目录号:430168,分别用于20和5ml培养基)
    2. 冰冷的Ficoll(Lymphoprep TM,密度1.077g/ml)(Abcys,目录号:1114545)
    3. 来自明串珠菌的硫酸葡聚糖钠盐,MW> 500,000(Sigma-Aldrich,目录号:D6001)
    4. 来自猪肠粘膜的肝素钠盐(Sigma-Aldrich,目录号:H3393)
    5. 来自水泡藻(Fucus vesiculosus)的岩藻依聚糖(Sigma-Aldrich,目录号:F5631)
    6. Hoechst 33342,10mg/ml水溶液(Life Technologies,Molecular Probes ,目录号:H-3570)。
    7. 无菌蒸馏水RNase DNase free(Life Technologies,  Gibco ,目录号:10977-035)
    8. 小鼠,大鼠或兔免疫和免疫前多克隆血清
    9. 单克隆抗体(mAbs)与玫瑰花形成iRBC的表面反应
    10. 来自生活在疟疾流行地区的个体的血清或血浆样品
    11. 来自疟疾非免疫个体的血清或血浆样品
      注意,所有血浆样品必须使用除肝素以外的抗凝剂(肝素在用于抗凝血活性的浓度下破坏多种菌株的玫瑰花结)收集。方便的抗凝剂是EDTA [乙二胺四乙酸]或柠檬酸钠)。 储存于-20°C或-80°C直至使用。
      注意:Abs是由患者自制或收集的。

设备

  1. 将5ml聚苯乙烯圆底无菌管(BD Biosciences,Falcon ,目录号:352058)
  2. 无菌实验室真空过滤器0.22μm(Stericup Filter Units Millipore,目录号:051446)
  3. 将无菌聚丙烯锥形离心管(15ml,BD Biosciences,Falcon,目录号:352097; 50ml,BD Biosciences,Falcon ,目录号:352098)
  4. 无菌一次性血清学1ml吸量管(Dominique Dutscher,目录号:999079)
  5. 专业手套(例如 Kimtech Sterling丁腈手套,Kimberley-Clark Professional,目录号:99211或Satin Plus手套Kimberley-Clark Professional,目录号:SP2330E或2220E)
  6. 1.4ml基质圆底非无菌管(Thermo Fisher Scientific,目录号:10630784)
  7. 显微镜载玻片(Thermo Fisher Scientific,目录号:10090431)和盖玻片22×22mm(Thermo Fisher Scientific,目录号:11728691)
  8. 用旋转叶片转子离心机(Thermo Fisher Scientific Heraeus Multifuge 3SR +离心机)
  9. 用A-4-38摆动转子离心机Eppendorf 5702(Thermo Fisher Scientific,目录号:05-400-318)
  10. 真空泵(ILMVAC Biovac,型号:104)
  11. 具有UV光和40x或100x放大率的荧光显微镜(Leica荧光显微镜DM5000B,HP Plan目镜10×22 507897,HCX Plan 100x油,HCXPL 40x PH2)
  12. 在37℃下在连续注视中的孵育器:5%O 2,5%CO 2和90%N 2(Thermo Fisher Scientific Binder孵育器 ,型号:CB210)
  13. 层流II类,A2型生物安全柜(例如Thermo Fisher Scientific HeraSafe,目录号:13-998-002)

程序

请注意,在处理过程中必须使用手套。

  1. 玫瑰花结的制备
    1. 准备玫瑰花形成寄生虫的寄生虫培养物(参见" 恶性疟原虫 Rosette formation assay ",第1节 和2)。准备足够的材料以执行每个测定一式三份:每个测定需要3×40μl富含玫瑰花的悬浮液,在2%血细胞比容(见1.i-j。)。这对应于初始的20μl培养物,6-8%寄生虫血症,
    2. 收获玫瑰花形成寄生虫的文化在成熟滋养体到裂殖体阶段(5-10%寄生虫血症)。避免晚期或分裂的裂殖体,形成松散的玫瑰花结
    3. 通过在冰冷的Ficoll上离心来富集玫瑰花结(参见" 恶性疟原虫 Rosette formation assay " - 第2部分)。玫瑰花结破坏测定最好用具有超过50%玫瑰花结的频率的寄生虫培养物进行。
    4. 通过在Fisher Scientific Heraeus Multifuge中在256×g(1,200rpm),室温下离心10分钟来旋转细胞。
    5. 使用连接到真空泵的1ml抽吸无菌移液管抽吸上清液。
    6. 在补充有10μg/ml Hoechst的4ml预热的CCM(参见Recipes)中轻轻地重新悬浮富含玫瑰花结的沉淀(一些玫瑰花结易于被简单的机械处理破坏,避免来回强烈吸液)。
    7. 轻轻混合并在37℃下在黑暗中在具有连续注视的培养箱中孵育10分钟(5%O 2,5%CO 2,90%N 2 )。
    8. 用CCM洗涤一次:在Fisher Scientific Heraeus Multifuge中在室温下加入900μlCCM,在256×g(1,200rpm)下离心3分钟。
    9. 取出上清液,并在CCM(2体积(体积)细胞沉淀+98体积CCM)中以2%血细胞比容重悬沉淀。 制剂的寄生虫血症通常在5%左右
    10. 在1.4个基质圆底管中分配40μl的玫瑰花结富集的RBC悬浮液的等分试样
    11. 离心机在Fisher Scientific Heraeus Multifuge中以178×g(1,000rpm)室温下离心5分钟。
    12. 弃去上清液,按第2节或第3节所述处理沉淀。
    13. 在CCM中制备肝素,硫酸葡聚糖,岩藻依聚糖或其他硫酸化聚糖的系列稀释液(0.1μg/ml - 1 mg/ml)。
    14. 在CCM中制备血清,血浆(起始稀释度1/5)或纯化的mAb或免疫球蛋白制备物的系列稀释液作为稀释剂。 包括阴性对照例如单独的CCM和非免疫血清(或Ig)和具有能够破坏玫瑰花结的血清或mAb的阳性对照。
  2. 用硫酸化聚糖进行解离测定
    1. 加入100μl稀释的硫酸化聚糖到玫瑰花状制剂(1-j部分)
    2. 在37°C孵育30分钟(使用培养箱,而不是水浴)
    3. 取出60微升培养基,并将沉淀的沉淀重悬在剩余的体积中
    4. 取10微升等分试样,将其放在显微镜载玻片上,并添加盖玻片
    5. 在显微镜下计数100-200个iRBC,并且评分iRBC参与玫瑰花结(即,具有结合两个或更多个未感染RBC的滋养体或裂殖体阶段)(图1)。
    图1.在用10μg/ml 硫酸葡聚糖孵育之前和之后的玫瑰花结离解测定。

    1. 计算玫瑰花结率=(不成熟阶段iRBC参与玫瑰花结/成熟阶段)×100。
    2. 计算解离速率=(无抑制剂的对照培养物的玫瑰花结速率 - 处理样品的玫瑰花结速率/无抑制剂的对照培养物的玫瑰花结速率)×100。
  3. 用免疫试剂进行解离测定
    1. 制备血清,血浆(起始稀释度1/5)或纯化的mAb或免疫球蛋白制备物的连续稀释液。 包括阴性对照例如单独的CCM和非免疫血清(或Ig)和具有能够破坏玫瑰花结的血清或mAb的阳性对照。
    2. 加入40μl稀释的血清或血浆到玫瑰花制备(1-n部分)
    3. 在37°C孵育30分钟(使用孵化器,而不是水浴)。
    4. 将沉淀的沉淀重悬在培养基中。
    5. 取10微升等分试样,把它放在显微镜载玻片上,并添加盖玻片。
    6. 在显微镜下计数,100-200 iRBCs和iRBC分数参与玫瑰花结。
    7. 计算玫瑰花结率=(不成熟阶段iRBC参与玫瑰花结/成熟阶段)×100。
    8. 计算解离速率=(用非免疫试剂孵育的对照培养物的玫瑰花结速率 - 处理样品的玫瑰状化速率/用非免疫试剂孵育的对照培养物的玫瑰形成速率)×100。 注意,在某些稀释度下,多克隆免疫试剂(和IgM mAb)倾向于破坏玫瑰花结并凝集iRBC。 免疫凝集物容易显现为密集包装的iRBC(参见图2)。 在较高稀释度下容易显现解离。


图2.孵育后获得的形成玫瑰花结的iRBC的凝集   与针对PfEMP1衍生的小鼠多克隆抗体 表面暴露结构域(稀释1/40)。 iRBC细胞核用Hoechst 33342染色,细胞用光A和UV B显微镜检查。

食谱

  1. 完全培养基(CCM,500ml)
    445ml RPMI 1640
    5ml次黄嘌呤溶液(终浓度100μM) 200μl庆大霉素溶液(终浓度20μg/ml) 50ml人AB血清+终浓度(终浓度10%) 使用0.22μm过滤器单元灭菌
    储存在4°C或-20°C长期储存
  2. 硫酸化聚糖的储备溶液 准备10mg/ml的硫酸化聚糖在无菌水(0.1g + 10ml水)中的溶液 使用0.22μm过滤器单元灭菌
    等分并存储在-80°C
    使用每个等分试样一次(不要冻融)

致谢

该方案改编自以下出版物:Vigan-Womas等人 (2008和2010)。 这项工作得到了Agence Nationale de la Recherche的合同ANR-07-MIME-021-0(www.agence-nationale-recherche.fr/)和第七个欧洲框架计划FP7/2007-2013(http: ://cordis.europa.eu/fp7/home_en.html)contract 242095,Evimalar。

参考文献

  1. Vigan-Womas,I.,Guillotte,M.,Le Scanf,C.,Igonet,S.,Petres,S.,Juillerat,A.,Badaut,C.,Nato,F.,Schneider,A.,Lavergne, A.,Contamin,H.,Tall,A.,Baril,L.,Bentley,GA和Mercereau-Puijalon,O。(2008)。 体内 和体外模型的恶性疟原虫玫瑰花结和由varO介导的自凝集,varO是编码频繁血清型的A组var基因。 Infect Immun 76(12):5565-5580。 br />
  2. Vigan-Womas,I.,Lokossou,A.,Guillotte,M.,Juillerat,A.,Bentley,G.,Garcia,A.,Mercereau-Puijalon,O.and Migot-Nabias, 对恶性疟原虫的体液反应 VarO玫瑰花结变体及其与保护的关联。 J 9:267。
  3. Vigan-Womas,I.,Guillotte,M.,Juillerat,A.,Vallieres,C.,Lewit-Bentley,A.,Tall,A.,Baril,L.,Bentley,GA和Mercereau-Puijalon, 2011)。 恶性疟原虫红细胞膜蛋白1的等位基因多样性需要变异特异性红细胞表面表位。

    /em> 6(1):e16544。
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引用:Guillotte-Blisnick, M., Mercereau-Puijalon, O. and Vigan-Womas, I. (2013). Plasmodium falciparum Rosette Disruption Assay. Bio-protocol 3(8): e411. DOI: 10.21769/BioProtoc.411.
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