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Plasmodium falciparum Rosette Formation Assay
恶性疟原虫莲座形成试验   

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Abstract

Rosetting, i.e. the capacity of red blood cells (iRBCs) infected with mature parasite stages to bind two or more uninfected red blood cells (RBCs) is a virulence factor of Plasmodium falciparum. This protocol describes an in vitro assay to monitor rosette formation by P. falciparum-infected red blood cells, including procedures for rosette enrichment, maintenance of rosetting phenotype and assays for rosetting with RBC labeled using lipophilic fluorescent probes.

Materials and Reagents

  1. Human red blood cells and parasitized red blood cells
    1. Fresh human red blood cells (RBCs) collected on anticoagulant, preferably citrate-phosphate-dextrose (CPD). Avoid use of heparin, as most rosettes are heparin-sensitive. If fresh RBCs (collected during the previous 24 h) are not available, use a sample stored for less than 2 weeks at 4 °C.
    2. P. falciparum parasitized red blood cells collected by veinipuncture from patients in the presence of CPD anticoagulant
    3. Laboratory lines of P. falciparum parasites: 3D7 clone obtained from MR4 or rosetting clones such as Palo Alto 89F5 VarO, IT4/R29, 3D7/PF13 (Vigan-Womas et al., 2011) or IT4/FCR3S1.2
  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. Hypoxanthine solution 100x (10 mM) (C.C.Pro, catalog number: Z-41-M )
    4. Gentamicin solution (50 mg/ml) (Sigma-Aldrich, catalog number: G1397 )
    5. Human AB+ serum (final concentration 10%)
    6. Complete culture medium (see Recipes)
  3. Other materials
    1. Non pyrogenic sterile polystyrene, Rectangular Canted Neck Cell Culture Flask with Plug Seal Cap (Corning Incorporated 75 cm2, catalog number: 430720 , 25 cm2, catalog number: 430168 , for 20 and 5 ml of culture medium, respectively)
    2. 5 ml polystyrene round-bottom sterile tube (BD Biosciences, Falcon®, catalog number: 352058 )
    3. Sterile laboratory vacuum filter 0.22 μm (Stericup® Filter Units Millipore, catalog number: 051446 )
    4. Sterile polypropylene conical centrifuge tubes (BD Biosciences, Falcon® 15 ml) (BD Biosciences,  catalog number: 352097 ), (Falcon 50 ml) (BD Biosciences, catalog number: 352098 )
    5. Sterile disposable serological 1 ml aspiration pipette (Dominique Dutscher, catalog number: 999079 )
    6. 1.4 ml matrix round bottom non sterile tubes (Thermo Fisher Scientific, catalog number: 10630784 )
    7. 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 )
    8. Microscope glass slides (Thermo Fisher Scientific, catalog number: 10090431 ) and cover slips 22 x 22 mm (Thermo Fisher Scientific, catalog number: 11728691 )
    9. Ice cold Ficoll (LymphoprepTM, density 1.077 g/ml) (Abcys, catalog number: 1114545 )
    10. Dextran sulfate sodium salt from Leuconostoc spp, MW > 500,000 (Sigma-Aldrich, catalog number: D6001 )
    11. Heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3393 )
    12. Hoechst 33342, 10 mg/ml solution in water (Molecular Probes, catalog number: H-3570 )
    13. PKH67 green fluorescent cell linker (Sigma-Aldrich, catalog number: MINI-67 )
    14. PKH26 red fluorescent cell linker (Sigma-Aldrich, catalog number: MINI-26 )
    15. Foetal bovine serum (Sigma-Aldrich, catalog number: F2442 )
    16. Sterile distilled water RNase DNase free (Life Technologies, Gibco®, catalog number: 10977-035 )
    17. Giemsa staining reagent: Giemsa R solution (RAL Diagnostics, catalog number: 320310-0500 )
    18. Giemsa buffer tablet pH 7.2 (Merck KGaA, catalog number: 1.09468.0100 )
    19. Methanol GPR Rectapur BDH Proloabo (catalog number: 20846.292 )
    20. Sterile distilled water
    21. Stock solutions of heparin or dextran sulfate (see Recipes)

Equipment

  1. Centrifuge with a swing bucket rotor (Thermo Fisher Scientific Heraeus Multifuge 3SR+ centrifuge )
  2. Centrifuge Eppendorf 5702 with an A-4-38 swing bucket rotor (Thermo Fisher Scientific, catalog number: 05-400-318)
  3. Vacuum pump (ILMVAC Biovac, modle: 104 )
  4. 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)
  5. Incubator at 37 °C in continuous gazing: 5% O2, 5% CO2 and 90% N2 (Thermo Fisher Scientific Binder incubator, model: CB210 )
  6. Laminar flow class II, type A2 biological safety cabinet (Fisher Scientific HeraSafe, catalog number: 13-998-002 )
  7. VarioMACSTM magnetic separator (Miltenyi Biotec)
  8. MACS separation CS columns (Miltenyi Biotec, catalog number: 130-041-305 )
  9. Chemistry Hood

Procedure

Use disposable professional gloves throughout.

  1. In vitro culture of Plasmodium falciparum erythrocytic stages
    For all details concerning the general method for in vitro culture of P. falciparum blood stage parasites and assessment of parasitaemia by blood smear see (Moll et al., 2008a; Alexander et al., 2012).
    1. Cultivate Plasmodium falciparum parasites in fresh human O+ or A+ erythrocytes at 5% haematocrit, in CCM (see recipes section), under continuous gazing (5% O2, 5% CO2 and 90% N2) at 37 °C. The gas mixture may also be introduced by blowing gas for 20 sec in the culture flask. Close the flask tightly thereafter. The Palo Alto 89F5 VarO, IT4/R29, 3D7/PF13 or IT4/FCR3S1.2 rosette-forming clones can be used as positive controls, as they have a high proportion of rosette-forming parasites. Note that the percentage of rosette-forming parasites in clinical samples is quite variable.
    2. Change medium every day: The flask is gently put under the laminar flow safety cabinet avoiding disturbing the sedimented red blood cells, after 15 min, the medium (supernatant) is carefully aspirated with a sterile disposable serological 1 ml aspiration pipette (devoid of cotton plug) connected to a vacuum pump.
    3. Count parasitemia daily. Prepare a blood smear and fix the cells by pouring pure methanol onto the slide.– Important note: Handle methanol under a chemistry hood, collect waste store it under a chemistry hood and use ad-hoc waste disposal circuit. Prepare a Giemsa-stained smear of methanol-fixed red cells by immersing the slide in a 5% Giemsa solution in buffer pH 7.2 prepared with 1 tablet of buffer Merck 1.09468.0100 in 1 L sterile distilled water. Stain for 10 min at room temperature. Rinse with buffer and examine under a microscope.
    4. Do not allow parasites to grow at too high or too low parasitaemia (up to 10% and less than 0.5%) otherwise they collapse (in practice, dilution is done every 4th day approximately)
      Note: Rosette formation is dependent on human serum components - practically speaking presence of human serum is needed to form rosettes. For each rosetting line or isolate, the exact concentration of serum needed should be determined. Usually, it is above 5%. Some commercial serum batches do not sustain rosette formation. Test the batch of sera with positive control lines, such as Palo Alto 89F5 VarO, IT4/R29, 3D7/PF13 (Vigan-Womas et al., 2011) or IT4/FCR3S1.2.

  2. Rosette enrichment on ice-cold Ficoll
    Centrifugation on ice-cold Ficoll enriches for rosette-forming iRBCs and mitigates the progressive loss of the phenotype resulting from antigenic variation. It can be done with cultures of rosetting parasites (e.g. Palo Alto 89F5 VarO, IT4/R29, 3D7/PF13 or FCR3S1.2) or used to enrich for rosette-forming iRBCs from unselected lines or clinical samples. Starting from a standard (unselected) long-term in vitro P. falciparum culture in which no or few rosettes are initially detected, enrichment twice to three times a week during successive 8-12 weeks allows progressive increase of the rosetting rate to approx. 10% (Vigan-Womas et al., 2011).
    1. Harvest in vitro culture at mature stages (trophozoite - young schizont stages, 5 to 8% parasitaemia) in a conical Falcon centrifugation tube.
    2. Centrifuge the culture, 10 min at 256 x g (1,200 rpm) in the Fisher Scientific Heraeus Multifuge at room temperature.
    3. Discard the supernatant.
    4. Resuspend the pellet in prewarmed (37 °C) CCM, e.g. add 11 ml CCM to 1 ml pellet.
    5. Dispense 2 ml ice-cold Ficoll in a 5 ml polystyrene round-bottom sterile tube (prepare six tubes for 1 ml cell pellet).
    6. Carefully overlay 2 ml RBC suspension (step d) on top of the Ficoll (step e).
    7. Centrifuge immediately in an Eppendorf Centrifuge 5702 for 30 sec, at 900 x g (2,400 rpm), room temperature. Do not use brake to decelerate. Rosette-forming parasites sediment with uninfected RBCs in the small pellet at the bottom of the tube.
    8. Quickly aspirate the supernatant using a 1 ml aspiration sterile pipette connected to a vacuum pump.
    9. Resuspend and pool the 6 pellets containing rosette-forming parasites in 4 ml prewarmed CCM.
    10. Check the rosetting rate (see 3).
    11. Repeat the procedure routinely twice a week.

  3. Assessment of the rosetting rate
    1. In a 1.4 ml matrix round bottom tube, add 100 μl of CCM containing 10 μg/ml Hoechst 33342 and 1-2 μl of cell pellet under study.
    2. Mix gently and incubate for 10 min at 37 °C in the dark.
    3. Add 900 μl CCM.
    4. Centrifuge 3 min at 256 x g (1,200 rpm), room temperature in the Fisher Scientific Heraeus Multifuge.
    5. Remove the supernatant and resuspend the pellet in 40 μl prewarmed CCM.
    6. Place a 10 μl parasite suspension onto a microscope glass slide and cover with a coverslip.
    7. Examine the sample under the microscope using a 40x or 100x objective. In case of Hoechst 33342-stained samples, use a fluorescence microscope and use both UV and bright-field light to visualise both iRBCs and RBC (Figure 1).


      Figure 1. Visualisation of rosettes after enrichment on Ice-cold Ficoll. A. Light microscopy. B. UV and light microscopy of Hoechst 33342-stained preparations. C. Giemsa stained smear.

    1. Count 100-200 iRBCs and score the number of iRBCs engaged in rosettes (i.e. trophozoite or schizont stages having bound two or more uninfected RBC).
    2. Calculate the rosetting rate = (No. mature stage iRBC engaged in rosettes/No. mature stages) x 100.

  4. Selection of mature iRBCs (trophozoites to schizont) by using magnetic cell sorting (MACS).
    1. Harvest in vitro culture of rosettes forming parasites at mature trophozoite to schizont stages.
    2. Enrich for rosettes on ice-cold Ficoll (as described in section 2).
    3. Prepare 10 μg/ml heparin or dextran sulphate in CCM and add 5 ml to the cell preparation to dissociate rosettes.
    4. Incubate 30 min at 37 °C (use an incubator with continuous gazing rather than water-bath).
    5. Check that all rosettes are disrupted by microscopic examination of an aliquot (see section 3).
    6. Place a CS column in the magnetic field of the VarioMACS magnetic separator and fill the column with 10 μg/ml heparin or dextran sulphate in CCM.
    7. Load the rosette-disrupted suspension on the column. Haemozoin-containing mature trophozoite and schizont stages are retained by the magnetic field.
    8. Wash twice the column with 10 ml 10 μg/ml heparin or dextran sulphate in CCM.
    9. Disconnect the column from the magnetic field and put it onto a 15 ml Falcon conical tube.
    10. Add 10 ml 10 μg/ml Heparin or Dextran sulphate in CCM on top of the column.
    11. Elute the retained iRBCs by flushing out the column using a 10 ml syringe filled with air.
    12. Centrifuge 10 min, 256 x g (1,200 rpm) in the Fisher Scientific Heraeus Multifuge at room temperature.
    13. Wash twice the pellet with RPMI medium supplemented with 1-2% Human AB+ serum (serum needed to avoid agglutination of iRBCs).
    14. Resuspend the pellet at 2% haematocrit in RPMI medium supplemented with 1-2% Human AB+ serum.
    15. Check parasitaemia of the purified iRBCs using a Giemsa-stained smear. The percentage of iRBCs should be > 90%.
      For additional experimental details concerning MACS purification of iRBCs with mature P. falciparum stages see (Moll et al. 2008b).

  5. Rosette formation assay with RBCs labeled using lipophilic fluorescent probes
    Analysis of rosette-forming iRBC preference can be done by incubating various ratios of differentially labeled uninfected RBCs with iRBCs obtained from dissociated rosettes and concentrated by magnetic cell sorting (MACS) (Vigan-Womas et al., 2012).
    1. Red blood cells labeling with PKH dyes
      Perform all steps at room temperature (21 °C to 25 °C)
      1. Place approximately 5 x 107 RBCs (i.e. 50 μl pelleted red cells or 500 μl red cells at 10% haematocrit in RPMI or CCM) in a 15 ml conical bottom polypropylene centrifuge tube.
      2. Add 5 ml RPMI 1640 medium (without serum).
      3. Centrifuge at 256 x g (1,200 rpm) in the Fisher Scientific Heraeus Multifuge, 5-10 min, room temperature.
      4. Carefully aspirate the supernatant, leaving no more than 25 μl supernatant on top of the pellet.
      5. Prepare a 1 μM PKH solution in diluent C provided by the manufacturer.
      6. Resuspend the RBC pellet in 500 μl diluent C by pipetting (do no vortex).
      7. Add 500 μl 1 μM PKH and immediately mix the sample by pipetting.
      8. Incubate 3 min at room temperature. Periodically invert the tube gently.
      9. Add 1 ml foetal bovine serum to stop the staining.
      10. Mix gently by inversion and incubate 1 min at room temperature.
      11. Add 2 ml CCM.
      12. Centrifuge at 256 x g (1,200 rpm) in the Fisher Scientific Heraeus Multifuge, 10 min, room temperature.
      13. Remove the supernatant and transfer the cell pellet in a new 15 ml conical tube.
      14. Wash twice with 10 ml CCM followed by centrifugation at 256 x g (1,200 rpm) in the Fisher Scientific Heraeus Multifuge, 10 min, room temperature.
      15. Resuspend the cell pellet at 2% haematocrit in RPMI medium, supplemented with 1-2% Human AB+ serum.
      16. Examine cell staining using fluorescence microscopy.
    2. Rosette formation with PKH-labeled RBCs
      1. Use the procedure described in section 4 to obtain a pure (or highly enriched) preparation of iRBCs from rosetting parasites.
      2. Label iRBCs with 10 μg/ml Hoechst 33342 (see section 3).
      3. Resuspend the pellet at 2% haematocrit in RPMI medium supplemented with 1-2% Human AB+ serum.
      4. Dispense 40 μl iRBCs aliquots in 1.4 matrix round bottom tubes.
      5. Centrifuge at 178 x g (1,000 rpm) in the Fisher Scientific Heraeus Multifuge, 5 min, at room temperature and aspirate the supernatant.
      6. Prepare tubes containing varying ratios of PKH 67- or PKH26-labeled RBCs in RPMI medium with 1-2% Human AB+ serum (see section 5-a).
      7. Add 100 μl PKH 67- or PKH26-labeled RBCs to iRBCs pellet from step v.
      8. Mix and wash twice the RBC/iRBC suspension with 900 μl CCM followed by centrifugation 5 min at 178 x g (1,000 rpm) in the Fisher Scientific Heraeus Multifuge, room temperature.
      9. Discard the supernatant.
      10. Resuspend the RBC/iRBC pellet in 200 μl CCM.
      11. Incubate for 1 h, at 37 °C (use an incubator rather than water-bath).
      12. Discard the supernatant and resuspend the pellet in 40 μl CCM.
      13. Place a 10 μl suspension onto a microscope glass slide and cover with a coverslip.
      14. Count the percentage of rosettes using a fluorescence microscope.
      15. Record the number of PKH 67- or PKH26-labeled RBCs engaged in rosettes (Figure 2).


        Figure 2: Rosette formation of VarO-iRBC with PKH-labeled recipient RBCs. A. Rosette formed upon incubation with PKH 67-labeled O+ RBCs. B. Rosette formed upon incubation with PKH 26-labeled A+ RBCs. C. Rosette formed upon incubation with a 1:1 mixture of PKH 67-labeled O+ and PKH 26-labeled A+ RBCs

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 heparin or dextran sulfate
    Prepare a 10 mg/ml solution of heparin or dextran sulphate 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. (2011) and Vigan-Womas et al. (2012). 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. Kirsten, M., Inger, L., Hedvig, P., Artur, S., Mats, W. (2008a) Methods in Malaria Research, MRA/ ATCC and BioMalPar. Fifth Edition, section I, p. 4-7.
  2. Kirsten, M., Inger, L., Hedvig, P., Artur, S., Mats, W. (2008b) Methods in Malaria Research, MRA/ ATCC and BioMalPar. Fifth Edition, section IV p 31-44. 
  3. Kirsten, M., Inger, L., Hedvig, P., Artur, S., Mats, W. (2008b) Methods in Malaria Research, Fifth Edition, section IV p 31-44, MRA/ ATCC and BioMalPar (www.biomalpar.org/updatedMethods_In_Malaria_Research_5thedition.pdf).
  4. Maier, A. G. and Rug, M. (2013). In vitro Culturing Plasmodium falciparum Erythrocytic Stages. Methods Mol Biol 923: 3-15.
  5. 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.
  6. Vigan-Womas, I., Guillotte, M., Juillerat, A., Hessel, A., Raynal, B., England, P., Cohen, J. H., Bertrand, O., Peyrard, T., Bentley, G. A., Lewit-Bentley, A. and Mercereau-Puijalon, O. (2012). Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting. PLoS Pathog 8(7): e1002781.

简介

Rosetting,即感染成熟寄生虫阶段以结合两个或更多个未感染的红细胞(RBC)的红细胞(iRBC)的能力是恶性疟原虫的毒力因子。 。 该方案描述了用于通过EM监测玫瑰花结形成的体外试验。 包括玫瑰花结富集程序,玫瑰花结表型的维持和用亲脂性荧光探针标记的RBC形成玫瑰花结的测定法。

材料和试剂

  1. 人红细胞和寄生的红细胞
    1. 在抗凝血剂,优选柠檬酸盐 - 磷酸盐 - 葡萄糖(CPD)上收集新鲜人红细胞(RBC)。 避免使用肝素,因为大多数玫瑰花结对肝素敏感。 如果无法获得新鲜RBC(在前24小时内收集),请使用在4°C下储存少于2周的样品。
    2. p。 在CPD抗凝血剂存在下通过静脉穿刺从患者收集的恶性疟原虫寄生的红细胞
    3. 实验室线。 恶性疟原虫寄生虫:从MR4或玫瑰花结克隆例如Palo Alto 89F5ValO,IT4/R29,3D7/PF13(Vigan-Womas等人,2011)或IT4/FCR3S1获得的3D7克隆 .2
  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. 次黄嘌呤溶液100x(10mM)(C.C.Pro,目录号:Z-41-M)
    4. 庆大霉素溶液(50mg/ml)(Sigma-Aldrich,目录号:G1397)
    5. 人AB血清(终浓度10%)
    6. 完全培养基(见配方)
  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细胞培养瓶(Corning Incorporated 75cm 2,目录号:430720,25cm 2,目录号:430168, 分别为20和5ml培养基)
    2. 5ml聚苯乙烯圆底无菌管(BD Biosciences,Falcon ,目录号:352058)。
    3. 无菌实验室真空过滤器0.22μm(Stericup Filter Units Millipore,目录号:051446)
    4. 将无菌聚丙烯锥形离心管(BD Biosciences,Falcon 15ml)(BD Biosciences,目录号:352097),(Falcon 50ml)(BD Biosciences,目录号:352098) >
    5. 无菌一次性血清学1ml吸量管(Dominique Dutscher,目录号:999079)
    6. 1.4ml基质圆底非无菌管(Thermo Fisher Scientific,目录号:10630784)
    7. 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)
    8. 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)
    9. 冰冷的Ficoll(Lymphoprep ,密度为1.077g/ml )(Abcys,目录号:1114545)
    10. 来自明串珠菌的硫酸葡聚糖钠盐,MW> 500,000(Sigma-Aldrich,目录号:D6001)
    11. 来自猪肠粘膜的肝素钠盐(Sigma-Aldrich,目录号:H3393)
    12. Hoechst 33342,10mg/ml的水溶液(Molecular Probes,目录号:H-3570)
    13. PKH67绿色荧光细胞接头(Sigma-Aldrich,目录号:MINI-67)
    14. PKH26红色荧光细胞接头(Sigma-Aldrich,目录号:MINI-26)
    15. 胎牛血清(Sigma-Aldrich,目录号:F2442)
    16. 无菌蒸馏水RNase DNase free(Life Technologies,  Gibco ,目录号:10977-035)
    17. Giemsa染色试剂:Giemsa R溶液(RAL Diagnostics,目录号:320310-0500)
    18. Giemsa缓冲片pH7.2(Merck KGaA,目录号:1.09468.0100)
    19. 甲醇GPR Rectapur BDH Proloabo(目录号:20846.292)
    20. 无菌蒸馏水
    21. 肝素或硫酸葡聚糖储备液(见配方)

设备

  1. 用旋转叶片转子离心机(Thermo Fisher Scientific Heraeus Multifuge 3SR +离心机)
  2. 用A-4-38摆动转子离心机离心Eppendorf 5702(Thermo Fisher Scientific,目录号:05-400-318)
  3. 真空泵(ILMVAC Biovac,型号:104)
  4. 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 40×PH2)
  5. 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)
  6. 层流II类,A2型生物安全柜(Fisher Scientific HeraSafe,目录号:13-998-002)
  7. VarioMACS TM磁性分离器(Miltenyi Biotec)
  8. MACS分离CS柱(Miltenyi Biotec,目录号:130-041-305)
  9. 化学罩

程序

使用一次性专业手套。

  1. 恶性疟原虫红/红细胞阶段的体外培养
    关于用于体外培养P的一般方法的所有细节。恶性疟原虫血液阶段寄生虫和通过血涂片评估寄生虫血症(参见(Moll等人,2008a; Alexander等人,2012)。
    1. 在连续注视下,在CCM(参见配方部分)中以5%血细胞比容在新鲜人O sup/+/sup /或A sup + +红细胞中培养镰状疟原虫(5%O 2,5%CO 2和90%N 2)。气体混合物也可以通过在培养瓶中吹入气体20秒来引入。然后紧紧关闭烧瓶。 Palo Alto 89F5VolO,IT4/R29,3D7/PF13或IT4/FCR3S1.2形成玫瑰花结的克隆可用作阳性对照,因为它们具有高比例的形成玫瑰花结的寄生虫。注意,临床样品中玫瑰花结形成寄生虫的百分比是相当可变的。
    2. 每天更换培养基:将烧瓶轻轻置于层流安全柜下,避免干扰沉积的红细胞,15分钟后,用无菌的一次性血清学1ml吸移管(无棉塞)小心吸出培养基(上清液) )连接到真空泵。
    3. 每日计数寄生虫血症。准备血液涂片,通过将纯甲醇倒在载玻片上固定细胞。 - 重要注意事项:在化学罩下处理甲醇,收集废物在化学罩下存储,并使用特殊废物处理回路。通过将载玻片浸没在1%无菌蒸馏水中的1片缓冲液Merck 1.09468.0100制备的5%吉姆萨溶液缓冲液pH7.2中制备甲醇固定的红细胞的吉姆萨染色的涂片。在室温下染色10分钟。用缓冲液冲洗,并在显微镜下检查
    4. 不要让寄生虫生长在太高或太低的寄生虫血症(高达10%和低于0.5%),否则它们会崩溃(实际上,每4天大约稀释一次)
      注意:玫瑰花结形成依赖于人血清成分 - 实际上,需要存在人血清以形成玫瑰花结。对于每个玫瑰花结线或分离物,应确定所需血清的确切浓度。通常,高于5%。一些商业血清批次不维持玫瑰花结形成。使用阳性对照线(例如Palo Alto 89F5ValO,IT4/R29,3D7/PF13(Vigan-Womas等人,2011)或IT4/FCR3S1.2)测试该批血清。
  2. 在冰冷的Ficoll上的玫瑰花结富集
    在冰冷的Ficoll上离心富集形成玫瑰花结的iRBC,并减轻由抗原变异引起的表型的进行性损失。它可以用玫瑰花结寄生虫的培养物(例如Palo Alto 89F5 VarO,IT4/R29,3D7/PF13或FCR3S1.2)进行,或用于富集来自未选择的品系或临床样品的玫瑰花结形成iRBC 。从标准(未选择的)长期体外 开始。最初检测到少数玫瑰花结的恶性疟原虫培养物,在连续8-12周期间每周富集两次至三次,允许玫瑰花结速率逐渐增加至大约50%。 10%(Vigan-Womas等人,2011)。
    1. 在锥形Falcon离心管中在成熟阶段(滋养体 - 幼年裂殖体阶段,5至8%寄生虫血症)收获体外培养物。
    2. 在Fisher Scientific Heraeus Multifuge中在室温下以256×g(1,200rpm)离心培养物10分钟。
    3. 弃去上清液。
    4. 在预热(37℃)CCM中重悬沉淀,例如。 加入11ml CCM至1ml沉淀。
    5. 在5ml聚苯乙烯圆底无菌管中分配2ml冰冷的Ficoll(制备6个管,用于1ml细胞沉淀)。
    6. 在Ficoll顶部小心地覆盖2ml RBC悬浮液(步骤d)(步骤e)。
    7. 立即在Eppendorf离心机5702中在900×g(2400rpm),室温下离心30秒。 不要使用制动器减速。 形成玫瑰花结的寄生虫在管的底部的小颗粒中沉积未感染的RBC
    8. 使用连接到真空泵的1ml吸出无菌吸液管快速吸出上清液。
    9. 重悬和汇集6粒含玫瑰花结形成寄生虫在4毫升预热的CCM。
    10. 检查玫瑰花结率(见3)。
    11. 每周重复该过程,每次两次。

  3. 玫瑰花结率的评价
    1. 在1.4ml基质圆底管中,加入100μl含有10μg/ml Hoechst 33342和1-2μl所研究的细胞沉淀的CCM。
    2. 轻轻混匀,在37℃,黑暗中孵育10分钟
    3. 加入900μlCCM
    4. 在Fisher Scientific Heraeus Multifuge中在室温下以256×g离心3分钟(1,200rpm)。
    5. 取出上清液,并将沉淀重悬在40μl预热的CCM中
    6. 将10微升寄生虫悬浮液放在显微镜载玻片上,盖上盖玻片。
    7. 在显微镜下使用40x或100x物镜检查样品。 在Hoechst 33342染色的样品的情况下,使用荧光显微镜并且使用UV和明场光以使两种iRBC 和RBC(图1)。


      图1.富集冰冷Ficoll后玫瑰花结的可视化。 A.光学显微镜。 B.Huechst 33342染色制剂的UV和光学显微镜检查。 C.吉姆萨染色涂片。

    1. 计数100-200个iRBC,并对参与玫瑰花结的iRBC(即滋养体或裂殖体阶段,结合两个或更多个未感染的RBC)的数目进行评分。
    2. 计算玫瑰花结率=(成熟阶段iRBC参与玫瑰花结/成熟阶段)×100。

  4. 通过使用磁性细胞分选(MACS)选择成熟iRBC(滋养体向裂殖体)。
    1. 收获在成熟滋养体至裂殖体阶段形成寄生虫的玫瑰花结的体外培养物。
    2. 在冰冷的Ficoll上富集莲座(如第2节所述)。
    3. 在CCM中制备10μg/ml的肝素或硫酸葡聚糖,并向细胞制备物中加入5ml以解离玫瑰花结。
    4. 在37°C孵育30分钟(使用连续凝视而不是水浴的孵化器)
    5. 检查所有玫瑰花瓣是否通过显微镜检查等分试样破坏(见第3部分)。
    6. 将CS柱置于VarioMACS磁性分离器的磁场中,并用CCM中的10μg/ml肝素或硫酸葡聚糖填充柱。
    7. 在柱上装载玫瑰花中断的悬浮液。 含有haemozoin的成熟滋养体和裂殖体阶段由磁场保留
    8. 用10 ml 10μg/ml 肝素或硫酸葡聚糖在CCM中洗涤两次柱子。
    9. 从磁场上断开色谱柱,并将其放在15毫升Falcon锥形管上
    10. 在柱顶部的CCM中加入10ml10μg/ml的肝素或硫酸葡聚糖。
    11. 通过使用填充有空气的10ml注射器冲洗柱来洗脱保留的iRBC。
    12. 在Fisher Scientific Heraeus Multifuge中在室温下离心10分钟,256×g(1,200rpm)。
    13. 用补充有1-2%人AB血清(避免iRBC凝集所需的血清)的RPMI培养基洗涤沉淀两次。
    14. 在补充有1-2%人AB血清的RPMI培养基中以2%血细胞比容重悬沉淀物。
    15. 使用吉姆萨染色的涂片检查纯化的iRBC的寄生虫血症。 iRBC的百分比应该> 90%。
      关于有成熟P的iRBC的MACS纯化的其它实验细节。 falciparum 阶段(Moll等人,2008b)。

  5. 使用亲脂性荧光探针标记的RBC的玫瑰花形成测定 形成玫瑰花结形成iRBC优选的分析可以通过将各种比率的差异标记的未感染RBC与通过解离玫瑰花结获得的iRBC进行孵育并通过磁性细胞分选(MACS)进行浓缩来进行(Vigan-Womas等人,2012 )。
    1. 红细胞用PKH染料标记
      在室温(21°C至25°C)下执行所有步骤
      1. 在15ml锥形底部聚丙烯离心机中放置大约5×10 7个RBC(即50μl沉淀红细胞或500μl红细胞,在RPMI或CCM中10%血细胞比容) 管
      2. 加入5ml RPMI 1640培养基(无血清)
      3. 在Fisher Scientific Heraeus Multifuge中在256×g离心(1,200rpm),在室温下5-10分钟。
      4. 小心吸出上清液,在沉淀上留下不超过25μl上清液。
      5. 在制造商提供的稀释剂C中制备1μMPKH溶液。
      6. 通过移液(不旋涡)将RBC沉淀重悬在500μl稀释液C中
      7. 加入500μl1μMPKH,立即通过移液混合样品。
      8. 在室温下孵育3分钟。 轻轻周期性倒转管。
      9. 加入1ml胎牛血清以终止染色
      10. 通过颠倒轻轻混合,并在室温下孵育1分钟。
      11. 加入2ml CCM。
      12. 在Fisher Scientific Heraeus Multifuge中,在室温下10分钟,以256×g离心(1,200rpm)。
      13. 除去上清液,并在新的15毫升锥形管中转移细胞沉淀。
      14. 用10ml CCM洗涤两次,然后在Fisher Scientific Heraeus Multifuge中在256×g(1,200rpm),室温下10分钟离心。
      15. 在补充有1-2%人AB血清的RPMI培养基中以2%血细胞比容重悬细胞沉淀。
      16. 使用荧光显微镜检查细胞染色
    2. 用PKH标记的RBCs形成玫瑰花结
      1. 使用第4节中描述的程序从玫瑰花结寄生虫获得纯的(或高度富集的)iRBC制剂。
      2. 用10μg/ml Hoechst 33342标记iRBC(参见第3部分)。
      3. 在补充有1-2%人AB血清的RPMI培养基中以2%血细胞比容重悬沉淀物。
      4. 在140个基质圆底管中分配40μliRBC等分试样
      5. 在Fisher Scientific Heraeus Multifuge中在178×g(1,000rpm)下在室温下离心5分钟,并抽吸上清液。
      6. 准备在含有1-2%人AB血清的RPMI培养基中含有不同比例的PKH 67或PKH26标记的RBC的管(参见第5-a部分)。
      7. 向步骤v。的iRBCs沉淀中加入100μlPKH 67或PKH26标记的RBC
      8. 混合并用900μlCCM洗涤两次RBC/iRBC悬浮液,然后在Fisher Scientific Heraeus Multifuge,室温下以178×g(1,000rpm)离心5分钟。
      9. 弃去上清液。
      10. 重悬在RBC/iRBC沉淀在200微升CCM。
      11. 孵育1小时,在37°C(使用孵化器,而不是水浴)
      12. 弃去上清液,并在40μlCCM中重悬沉淀。
      13. 将10μl悬浮液放在显微镜载玻片上,盖上盖玻片。
      14. 使用荧光显微镜计算玫瑰花结的百分比。
      15. 记录参与玫瑰花结的PKH 67或PKH26标记的RBC的数量(图2)。


        图2:具有PKH标记的受体RBC的VarO-iRBC的玫瑰花结形成。 A.与PKH 67标记的O + RBCs孵育形成玫瑰花结。 B.与PKH 26标记的A + RBCs孵育形成的玫瑰花结。 C.用PKH 67标记的O +和PKH 26标记的A + RBC的1:1混合物孵育形成玫瑰花

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. 肝素或葡聚糖硫酸盐的储备溶液 制备10mg/ml的肝素或硫酸葡聚糖溶液在无菌水(0.1g + 10ml水中)中,使用0.22μm过滤器单元消毒,等分并储存在-80℃下,每次使用一次(不冻融 )

致谢

该方案改编自以下出版物:Vigan-Womas等人(2011)和Vigan-Womas等人(2012)。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. Kirsten,M.,Inger,L.,Hedvig,P.,Artur,S.,Mats,W。(2008a)Methods in Malaria Research,MRA/ATCC and BioMalPar。第五版,第一节, 4-7。
  2. Kirsten,M.,Inger,L.,Hedvig,P.,Artur,S.,Mats,W。(2008b)Methods in Malaria Research,MRA/ATCC和BioMalPar。第五版,第四部分,第31-44页。
  3. Kirsten,M.,Inger,L.,Hedvig,P.,Artur,S.,Mats, Research,Fifth Edition,section IV p 31-44,MRA/ATCC and BioMalPar(www.biomalpar.org/updatedMethods_In_Malaria_Research_5thedition.pdf)。
  4. Maier,A.G。和Rug,M。(2013)。体外培养恶性疟原虫红细胞期。 Methods Mol Biol 923:3-15。
  5. Vigan-Womas,I.,Guillotte,M.,Juillerat,A.,Vallieres,C.,Lewit-Bentley,A.,Tall,A.,Baril,L.,Bentley,GA和Mercereau-Puijalon, 2011)。 镰状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。 PLoS One 6(1):e16544。
  6. Vigan-Womas,I.,Guillotte,M.,Juillerat,A.,Hessel,A.,Raynal,B.,England,P.,Cohen,JH,Bertrand,O.,Peyrard,T.,Bentley, Lewit-Bentley,A.和Mercereau-Puijalon,O。(2012)。 恶性疟原虫玫瑰花结的ABO血型依赖性的结构基础。 PLoS Pathog 8(7):e1002781。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Vigan-Womas, I., Guillotte-Blisnick, M. and Mercereau-Puijalon, O. (2013). Plasmodium falciparum Rosette Formation Assay. Bio-protocol 3(8): e412. DOI: 10.21769/BioProtoc.412.
  2. Vigan-Womas, I., Guillotte, M., Juillerat, A., Hessel, A., Raynal, B., England, P., Cohen, J. H., Bertrand, O., Peyrard, T., Bentley, G. A., Lewit-Bentley, A. and Mercereau-Puijalon, O. (2012). Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting. PLoS Pathog 8(7): e1002781.
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