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Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model
使用秀丽隐杆线虫作为模型测定小分子药物的驱肠虫活性   

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Abstract

This protocol proposes to use the nematode Caenorhabditis elegans as a model to screen and study the anthelmintic activity of natural and synthetic compounds and to observe their effects on the morphology and the ultrastructure of the helminths. Furthermore, C. elegans can be used to investigate the anthelmintic activity in embryonated eggs, larval stages and in the adults’ survival. As most current anthelmintics are not effective against all nematode life stages, this protocol can contribute to the identification of new alternatives to helminthic infections (Sant’Anna et al., 2016).

Keywords: C. elegans(秀丽隐杆线虫), Nematodes(线虫), Anthelmintic drugs(驱虫药), Chemotherapy(化学疗法)

Background

Caenorhabditis elegans is a model organism for parasite nematode research and an excellent system for the screening of compounds with potential anthelmintic activity, because it is inexpensive, readily available, and easy to work with. In addition, the use of C. elegans in assays to investigate nematode behavior, locomotion, reproduction and death is uncomplicated and reliable (Simpkin and Coles, 1981). The protocols for screening new compounds on C. elegans were first carried out in axenic liquid medium in deep well microscope slides (Tomlinson et al., 1985) or using the drugs added to melted modified NGM agar (Driscoll et al., 1989). These methods are not effective in drug screening as axenic cultures, containing low food supply, trigger the intra-uterine birth causing maternal death (endotokia matricida) (Lenaerts et al., 2008) and drugs added to melted agar can modify drug stability due to the high temperatures. In this protocol, we used 96-well plates with liquid medium supplied with Escherichia coli to evaluate each stage (eggs, L1-L2 larvae, L3-L4 larvae and adults) independently.

Materials and Reagents

  1. Transfer pipette
  2. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352095 )
  3. 96-well plate, flat bottom, polystyrene, 0.32 cm2, sterile. TPP tissue culture plates (Sigma-Aldrich, catalog number: Z707910 )
  4. Tissue culture dishes of polystyrene TPP- diam. 60 x 15 mm, surface area size 22.1 cm2 with NGM (Sigma-Aldrich, catalog number: Z707678 )
  5. C. elegans N2 strain
  6. Escherichia coli OP50 strain
  7. Drugs to screen
  8. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 795429 )
  9. Hypochlorite (NaClO) (Sigma-Aldrich, catalog number: 13440 )
  10. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  11. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S5136 )
  12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  13. Magnesium sulfate heptahydrate (MgSO4·7H2O), BioUltra ≥ 99.5% (KT) (Sigma-Aldrich, catalog number: 63138 )
  14. Potassium phosphate dibasic (K2HPO4), ACS reagent, ≥ 98% (Sigma-Aldrich, catalog number: P3786 )
  15. Cholesterol (Sigma-Aldrich, catalog number: C3045 )
  16. Ethanol (p.a., without additive, ≥ 99.8%) (Sigma-Aldrich, catalog number: 24102 )
    Note: This product has been discontinued.
  17. Citric acid monohydrate (ACS reagent, ≥ 99.0%) (Sigma-Aldrich, catalog number: C1909 )
  18. Tri-potassium citrate monohydrate (Sigma-Aldrich, catalog number: 6100-05-6 )
  19. Disodium EDTA (98.5-101.5%, BioUltra) (Sigma-Aldrich, catalog number: E1644 )
  20. Iron (II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: 215422 )
  21. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 203734 )
  22. Zinc sulfate heptahydrate (ZnSO4·7H2O) (BioReagent, suitable for cell culture) (Sigma-Aldrich, catalog number: 7446-20-0 )
  23. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (BioReagent, suitable for cell culture, ≥ 98%) (Sigma-Aldrich, catalog number: C8027 )
  24. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  25. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  26. Lysing solution (see Recipes)
  27. M9 buffer (1 L) (see Recipes)
  28. S medium (1 L) (see Recipes)

Equipment

  1. Clinical centrifuge (Thermo Fisher Scientific, catalog number: 22-029-416 )
  2. Inverted microscope (ZEISS, model: Axio Vert.A1 )
  3. Biochemical oxygen demand (BOD) incubator (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 37-20 )
  4. Micropipet, 100-1,000 μl volume (Nichipet Eco pipette, catalog number: Z710199 )
  5. Autoclave

Procedure

  1. Culture synchronization (adapted from Stiernagle, 2006)
    1. Begin the procedure with C. elegans plates containing nearly 500 gravid hermaphrodites. Add 5 ml of M9 buffer to the plate and gently stir the liquid with a pipette to dislodge the worms from the agar.
    2. Using a pipette, transfer the worms to a 15 ml sterile Falcon tube, centrifuge at 800 x g for 1 min. The worm pellet must be suspended in 3.5 ml total volume.
    3. Add 1.5 ml of lysing solution to the tube. Shake the tube gently for 5 min, manually, looking under a microscope to check if worms have disintegrated or not. Most of the worm bodies have dissolved, centrifuge at 800 x g for 1 min.
    4. Remove most of lysing solution without disturbing the egg pellet.
    5. Then, suspend the pellet in 5 ml of M9 buffer, centrifuge 1,000 x g for 5 min, remove the supernatant, add fresh M9 and repeat these steps 2 more times.
    6. The eggs obtained can be conducted in three different procedures in parallel.
      I, The eggs can be incubated with the drugs directly for 15 h at 20 °C.
      II, The eggs can be incubated at 20 °C for 15 h to produce larvae, which can be incubated at maximum for 24 h with the drugs, to avoid development in advanced stages.
      III, The eggs can be incubated at 20 °C for 3 days to produce adults. Adults can be incubated, at maximum, for 3 days with the drugs to avoid new adult generations. Figure 1 summarizes the culture synchronization and the use of the three different life stages of C. elegans – embryonic eggs, larvae and adults (steps B, C and D) to evaluate anthelmintic activity of drugs.



      Figure 1. Scheme summarizing the culture synchronization procedure and the use of, embryonic eggs, larvae and the adults (steps B, C and D) of Caenorhabditis elegans to evaluate anthelmintic activity of drugs

  2. Egg hatch assay
    1. After the synchronization procedure, transfer the eggs to a 96-well plate with a pipette.
    2. Add approximately 30 eggs per well in 200 µl of S medium.
    3. Count and identify the embryonic stages inside the eggs with an inverted microscope to observe the larval development.
      Note: The presence of the gastrula form is important to determine the initial pattern before treatment with the drugs and to verify if the lysing solution affected or not the eggs. The gastrula form can be identified according to Figure 2B.
    4. Add the different concentrations of drugs and incubate for 15 h at 20 °C in a BOD incubator.
    5. At the end of the incubation, the percentage of hatched and unhatched eggs and the L1 larvae will be determined for each of the different drug concentrations by light microscopy. Triplicates of six independent experiments should be performed.



      Figure 2. Phase contrast light microscopy images showing different C. elegans stages. A. Embryo in the first cleavage; B. Gastrula form; C. Embryo with comma form; D. Egg with a larva inside (the 3-fold stage indicates the complete larval development); E. L1 stage; F. Hermaphrodite, the vulva (v) can be observed; G. Vulva (v) and numerous eggs (e) in higher magnification; H. The hermaphrodite gonad (g) can be observed.

  3. Larval development assay
    1. After the synchronization procedure, put the eggs in microtubes containing S medium and E. coli under gentle agitation, for 15 h at 20 °C at the BOD incubator.
    2. After this time, collect the larvae at the first stages (L1/L2) (Figure 2E). Alternatively, the eggs can hatch in the absence of food as development will be arrested and larvae will stay as L1.
    3. Adjust the concentration of larvae to 20 larvae/50 μl in S medium.
    4. Incubate for 24 h at 20 °C in a 96- well plates containing S medium supplemented with E. coli and the different concentrations of the drugs to be analyzed. Triplicates of six independent experiments should be performed.

  4. Assays using adults
    1. After the synchronization procedure, transfer the eggs to NGM (Nematode growth medium) plates seeded with E. coli with a pipette. The eggs were incubated at 20 °C for three days in the BOD incubator to obtain most of the adult nematodes at the same age.
    2. Collect the adult worms with a transfer pipette by washing the NGM plates with 5 ml of M9 to dislodge the worms and centrifuge at 800 x g for 5 min in a Falcon tube. Remove the supernatant and wash the pellet in the same buffer (three times).
    3. Place thirty nematodes per well in a 96-well plate containing S medium 200 µl supplemented with E. coli. The bacteria can be autoclaved to avoid excessive growth during the period of incubation. We suggest 2 x 103 bacteria/ml approximately.
    4. Add the different concentrations of the drugs to be screened in the study. The drugs must be omitted in the wells used as negative controls.
    5. Incubate at 20 °C for 3 days in the BOD incubator. Three days should be the maximum period of incubation to avoid the presence of new generations of adults.
    6. After this period, the survival was evaluated by counting the live and dead worms by light microscopy considering the motility and the paralysis of the pharyngeal bulb and the total loss of motility with the occurrence of straight bodies as shown in Video 1. Use an inverted microscope to make these observations. Triplicates of six independent experiments should be performed.

      Video 1. Movie illustrating the motility and the paralysis of the pharyngeal bulb by phase contrast light microscopy with the occurrence of straight bodies of dead worms

Data analysis

  1. To evaluate the survival of adults and larvae and their motility, living worms must be counted by optical microscopy excluding the larvae stages, which hatched during the assay. The size and the presence of reproductive organs are used as criteria to follow the original adult population (Figures 2F-2H).
  2. In the egg hatch assay, the percentage of hatched and unhatched eggs and survival of L1 larvae should be determined for each different drug concentration (as in Sant’Anna et al., 2016).
  3. Kaplan Meyer tests can be used to analyze the survival of individuals along the treatment and the survival curves should be compared by the log rank test (Kaplan and Meier, 1958).

Notes

The absence or low concentration of cholesterol affects the embryos survival, because it is essential for the development of the oocytes. The S medium should contain adequate concentrations of cholesterol (Greenstein, 2005).

Recipes

  1. Lysing solution
    5 N NaOH
    1% hypochlorite
    Prepared fresh for each batch
  2. M9 buffer (1 L)
    3 g KH2PO4
    6 g Na2HPO4
    5 g NaCl
    0.25 g MgSO4·7H2O
    Autoclave and stock at 4 °C
  3. S medium (1 L)
    1. S basal
      5.85 g NaCl
      1 g K2HPO4
      6 g KH2PO4
      1 ml cholesterol (5 mg/ml in ethanol)
    2. 10 ml 1 M potassium citrate, pH 6
      20 g citric acid monohydrate
      293.5 g tri-potassium citrate monohydrate
      Add H2O to 1 L
      Sterilize by autoclaving
    3. 10 ml trace metals solution
      1.86 g disodium EDTA
      0.69 g FeSO4·7H2O
      0.2 g MnCl2·4H2O
      0.29 g ZnSO4·7H2O
      0.025 g CuSO4·5H2O
      Add H2O to 1 L
      Sterilize by autoclaving and keep protected from light
    4. 3 ml 1 M CaCl2
    5. 3 ml 1 M MgSO4
    Note: Manipulate the components under sterile conditions; do not autoclave the complete medium. All the recipes were adapted from Stiernagle (2006).

Acknowledgments

The authors thank the Caenorhabditis Genetics Center for donating the C. elegans wild type and the Brazilian agencies: Fundação de Coordenação de aperfeiçoamento de Pessoal de Nível Superior-CAPES, Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ for supporting this work.

References

  1. Driscoll, M., Dean, E., Reilly, E., Bergholz, E. and Chalfie, M. (1989). Genetic and molecular analysis of a Caenorhabditis elegans β-tubulin that conveys benzimidazole sensitivity. J Cell Biol 109(6 Pt 1): 2993-3003.
  2. Greenstein, D. (2005). Control of oocyte meiotic maturation and fertilization. WormBook: 1-12.
  3. Kaplan, E. L. and Meier, P. (1958). Nonparametric estimation from incomplete observations. J Amer Statist Assn 53(282): 457-481.
  4. Lenaerts, I., Walker, G. A., Van Hoorebeke, L., Gems, D. and Vanfleteren, J. R. (2008). Dietary restriction of Caenorhabditis elegans by axenic culture reflects nutritional requirement for constituents provided by metabolically active microbes. J Gerontol A Biol Sci Med Sci 63(3): 242-252.
  5. Sant’Anna, V., de Souza, W. and Vommaro, R. C. (2016). Anthelmintic effect of herbicidal dinitroanilines on the nematode model Caenorhabditis elegans. Exp Parasitol 167: 43-49.
  6. Simpkin, K. G. and Coles, G. C. (1981). The use of Caenorhabditis elegans for anthelmintic screening. J Chem Technol Biotechnol 31(1): 66-69.
  7. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook 2(11).
  8. Tomlinson, G., Albuquerque, C. A. and Woods, R. A. (1985). The effects of amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) on Caenorhabditis elegans. Eur J Pharmacol 113(2): 255-262.

简介

该方案建议使用线虫秀丽隐杆线虫作为模型来筛选和研究天然和合成化合物的驱虫活性,并观察其对趾甲的形态和超微结构的影响。此外,C。线虫可用于研究胚胎卵,幼虫期和成年人生存期的驱虫活性。由于目前的大多数驱虫药对所有的线虫生命阶段均无效,因此该方案可有助于确定蠕虫感染的新替代品(2016年Sant'Anna等人)。

背景 秀丽隐杆线虫是寄生线虫研究的典型生物,是具有潜在驱虫活性的化合物筛选的优良系统,因为它便宜,易于获得并且易于使用。另外,使用 C。线虫在调查线虫行为,运动,繁殖和死亡的测定中是不复杂和可靠的(Simpkin和Coles,1981)。用于筛选新化合物的方案。首先在深孔显微镜载玻片(Tomlinson等人,1985)中的无菌液体培养基中进行线虫,或使用加入熔融修饰的NGM琼脂中的药物(Driscoll等人,1989)。这些方法在药物筛选中无效,因为含有低食物供应的无菌培养物,引发子宫内出生导致产妇死亡(“内毒素”)(Lenaerts等人) 2008),添加到熔化琼脂中的药物可以改善由于高温导致的药物稳定性。在该方案中,我们使用96孔平板与大肠杆菌供应的液体培养基独立地评估每个阶段(卵,L1-L2幼虫,L3-L4幼虫和成年人)。

关键字:秀丽隐杆线虫, 线虫, 驱虫药, 化学疗法

材料和试剂

  1. 转移移液管
  2. 15 ml Falcon管(Corning,Falcon ®,目录号:352095)
  3. 96孔板,平底,聚苯乙烯,0.32厘米2,无菌。 TPP组织培养板(Sigma-Aldrich,目录号:Z707910)
  4. 聚苯乙烯TPP直径的组织培养皿。使用NGM(Sigma-Aldrich,目录号:Z707678)的60×15mm,表面积尺寸22.1cm 2
  5. C。线虫 N2株
  6. 大肠杆菌OP50株
  7. 药物要屏蔽
  8. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:795429)
  9. 次氯酸盐(NaClO)(Sigma-Aldrich,目录号:13440)
  10. 磷酸二氢钾(KH 2 O 3 PO 4)(Sigma-Aldrich,目录号:P5655)
  11. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S5136)
  12. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  13. 硫酸镁七水合物(MgSO 4·7H 2 O),BioUltra≥99.5%(KT)(Sigma-Aldrich,目录号:63138)
  14. 磷酸氢二钾(K 2 O 3 HPO 4),ACS试剂,≥98%(Sigma-Aldrich,目录号:P3786)
  15. 胆固醇(Sigma-Aldrich,目录号:C3045)
  16. 乙醇(p.a.,无添加剂,≥99.8%)(Sigma-Aldrich,目录号:24102)
    注意:本产品已停产。
  17. 柠檬酸一水合物(ACS试剂,≥99.0%)(Sigma-Aldrich,目录号:C1909)
  18. 柠檬酸三钾一水合物(Sigma-Aldrich,目录号:6100-05-6)
  19. EDTA二钠(98.5-101.5%,BioUltra)(Sigma-Aldrich,目录号:E1644)
  20. 硫酸铁(II)七水合物(FeSO 4·7H 2 O)(Sigma-Aldrich,目录号:215422)
  21. 四氢化锰(II)四水合物(MnCl 2·4H 2 O)(Sigma-Aldrich,目录号:203734)
  22. 硫酸锌七水合物(ZnSO 4·7H 2 O)(BioReagent,适用于细胞培养)(Sigma-Aldrich,目录号:7446-20-0) >
  23. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(BioReagent,适用于细胞培养,≥98%)(Sigma-Aldrich,目录号:C8027)
  24. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  25. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  26. 溶解液(参见食谱)
  27. M9缓冲液(1升)(见配方)
  28. S中(1 L)(见配方)

设备

  1. 临床离心机(Thermo Fisher Scientific,目录号:22-029-416)
  2. 倒置显微镜(ZEISS,型号:Axio Vert.A1)
  3. 生化需氧量(BOD)培养箱(Thermo Fisher Scientific,Fisher Scientific TM ,目录号:37-20)
  4. Micropipet,100-1,000μl体积(Nichipet Eco移液器,目录号:Z710199)
  5. 高压灭菌器

程序

  1. 文化同步(改编自Stiernagle,2006)
    1. 用含有近500个妊娠雌雄同体的 C.elegans 板开始手术。在板中加入5ml M9缓冲液,用移液管轻轻搅拌液体,以从琼脂中除去蠕虫。
    2. 使用移液管将蠕虫转移到15ml无菌Falcon管中,以800 x g离心1分钟。蠕虫颗粒必须以3.5ml总体积悬浮。
    3. 向管中加入1.5ml裂解溶液。轻轻摇动管子5分钟,手动观察,在显微镜下观察,检查蠕虫是否已经瓦解。大多数蠕虫体已经溶解,以800 x g离心1分钟。
    4. 去除大部分裂解溶液,而不会干扰蛋颗粒。
    5. 然后,将沉淀物悬浮在5ml的M9缓冲液中,离心1000×g 5分钟,除去上清液,加入新鲜的M9并重复这些步骤2次。
    6. 所获得的卵可以以三种不同的程序并行进行 我可以将蛋与药物在20℃下直接孵育15小时。
      II,可以将蛋在20℃孵育15小时以产生幼虫,最后可以用药物孵育24小时,以避免在晚期发育。
      III,蛋可以在20℃下孵育3天以产生成年人。成年人最多可以与药物一起孵育3天,以避免新生代。图1总结了文化同步和使用三个不同的生命阶段。线虫 - 胚胎卵,幼虫和成年人(步骤B,C和D)以评估药物的驱虫活性。



      图1.总结培养同步程序的方案,以及胚芽卵,幼虫和成年人(步骤B,C和D)的使用,以评估药物的驱虫活性强>

  2. 蛋孵卵测定
    1. 在同步程序之后,用移液管将蛋转移到96孔板上。
    2. 在200μlS培养基中每孔加入约30个鸡蛋。
    3. 用倒置显微镜计数和鉴定鸡蛋内的胚胎阶段,观察幼虫发育。
      注意:在用药物处理之前,确定原始形式的存在对于确定初始模式是重要的,并且验证裂解溶液是否影响了卵。可以根据图2B识别原肠形式
    4. 加入不同浓度的药物,并在BOD培养箱中于20℃孵育15小时。
    5. 在孵化结束时,通过光学显微镜检测每种不同药物浓度的阴影和未孵化的蛋和L1幼虫的百分数。应进行六次独立实验的重复。



      图2.显示不同的C的相差光学显微镜图像。线虫阶段。 A.胚胎在第一次切割; ; ula形C.用逗号表示的胚胎D.里面有一只幼虫的鸡蛋(3倍的阶段表示完全的幼虫发育); E. L1期;外阴(v)可以观察; G. Vulva(v)和许多鸡蛋(e)在更高的放大倍数; H.可以观察雌雄同体性腺(g)。

  3. 幼虫发育测定
    1. 在同步程序之后,在温和的搅拌下将蛋放入含有S培养基和大肠杆菌的微管中,在20℃,BOD培养箱中放置15小时。
    2. 此后,在第一阶段收集幼虫(L1/L2)(图2E)。或者,鸡蛋可以在没有食物的情况下孵化,因为发育将被逮捕,幼虫将保持为L1。
    3. 在S培养基中将幼虫的浓度调整为20只幼虫/50μl。
    4. 在含有补充有E的S培养基的96孔板中,在20℃下孵育24小时。大肠杆菌和不同浓度的待分析药物。应进行六次独立实验的重复。

  4. 使用成人的测定
    1. 在同步程序之后,将卵转移到用Em种植的NGM(线虫生长培养基)板上。大肠杆菌用移液管。将蛋在BOD培养箱中在20℃下孵育3天以获得大部分同龄成虫线虫。
    2. 通过用5ml M9洗涤NGM板收集成年蠕虫,以移除蠕虫,并在Falcon管中以800 x g离心5分钟。取出上清液,并在同一缓冲液(3次)中洗涤沉淀。
    3. 在含有补充有E的S培养基的96孔板中每孔放置三十根线虫。大肠杆菌。细菌可以进行高压灭菌,以避免在孵育期间过度生长。我们建议约2×10 3细菌/ml。
    4. 在研究中添加不同浓度的待筛选药物。在用作阴性对照的孔中必须省略药物。
    5. 在BOD培养箱中在20℃下孵育3天。三天应该是最大的孵化期,以避免新一代的成年人的存在。
    6. 在这一时期之后,通过光学显微镜计数生活和死亡蠕虫,通过视频1所示,考虑咽喉的运动性和瘫痪以及直体发生的总动员丧失。使用倒置显微镜做出这些观察。应进行六次独立实验的重复。

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      视频1.通过相位对比光显微镜观察咽部球茎的运动性和瘫痪的动画,发生死亡蠕虫的直体
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数据分析

  1. 为了评估成虫和幼虫的生存及其活力,活检蠕虫必须通过光学显微镜计数,不包括测定期间孵出的幼虫阶段。使用生殖器官的大小和存在作为遵循原始成年人口的标准(图2F-2H)。
  2. 在卵孵化测定中,应确定每种不同药物浓度的孵化和未孵化的卵的百分比和L1幼虫的存活率(如Sant'Anna等人,2016)。
  3. Kaplan Meyer检验可用于分析治疗期间个体的生存情况,生存曲线应通过对数秩检验进行比较(Kaplan和Meier,1958)。

笔记

胆固醇的缺乏或低浓度影响胚胎的存活,因为它对卵母细胞的发育至关重要。 S培养基应含有足够的胆固醇(Greenstein,2005)。

食谱

  1. 溶解液
    5 N NaOH
    1%次氯酸盐
    新鲜准备的每批批次
  2. M9缓冲区(1 L)
    3g KH 2 PO 4
    6g Na 2 HPO 4
    5克NaCl
    0.25g MgSO 4·7H 2 O→// 高压灭菌和储存在4°C
  3. S中(1升)
    1. S基础
      5.85克NaCl
      1 g K 2 HPO 4
      6g KH 2 PO 4
      1毫升胆固醇(5毫克/毫升乙醇)
    2. 10ml 1M柠檬酸钾,pH 6
      20克柠檬酸一水合物
      293.5g柠檬酸三钾一水合物
      将H 2 O添加到1 L
      通过高压灭菌消毒
    3. 10ml痕量金属溶液
      1.86g EDTA二钠盐 0.69g FeSO 4·7H 2 O -/
      0.2g MnCl 2·4H 2 O
      0.29g ZnSO 4·7H 2 O
      0.025g CuSO 4·5H 2 O
      将H 2 O添加到1 L
      通过高压灭菌消毒,保护免受光照
    4. 3ml 1M CaCl 2
    5. 3ml 1M MgSO 4
    注意:在无菌条件下操作组分;不要对整个介质进行高压灭菌。所有的食谱都是从Stiernagle(2006)改编而成。

致谢

作者感谢Caenorhabditis遗传中心捐赠了C。 elegans 野生型和巴西机构:FundaçãodeCoordinatorçãodeaperfeiçoamentode Pessoal deNívelSuperior-CAPES,Conselho Nacional de DesenvolvimentoCientíficoeTecnológico-CNPq和FundaçãoCarlos Chagas Filho de AmparoàPesquisa do Estado do Rio de Janeiro -FAPERJ支持这项工作。

参考文献

  1. Driscoll,M.,Dean,E.,Reilly,E.,Bergholz,E.和Chalfie,M。(1989)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/2592410"target ="_ blank">传达苯并咪唑敏感性的秀丽隐杆线虫β-微管蛋白的遗传和分子分析细胞生物学 109(6 Pt 1):2993-3003。
  2. Greenstein,D。(2005)。控制卵母细胞减数分裂成熟和受精。 WormBook :1-12。
  3. Kaplan,EL和Meier,P.(1958)。< a class ="ke-insertfile"href ="http://www.tandfonline.com/doi/abs/10.1080/01621459.1958.10501452"target ="_ blank ">来自不完整观察结果的非参数估计。美国统计学家协会53(282):457-481。
  4. Lenaerts,I.,Walker,GA,Van Hoorebeke,L.,Gems,D.and Vanfleteren,JR(2008)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/18375873"target ="_ blank">通过无菌培养的秀丽隐杆线虫饮食限制反映了代谢活性微生物提供的成分的营养需求。 A Biol Sci Med Sci 63(3):242-252。
  5. Sant'Anna,V.,de Souza,W.和Vommaro,RC(2016)。  除草二硝基苯胺对线虫模型秀丽隐杆线虫的启示效应 167 Parasitol 167:43-49。
  6. Simpkin,KG和Coles,GC(1981)。< a class ="ke-insertfile"href ="http://onlinelibrary.wiley.com/doi/10.1002/jctb.503310110/abstract"target ="_ blank" >使用秀丽隐杆线虫进行驱肠虫筛选。 J Chem Technol Biotechnol 31(1):66-69。
  7. Stiernagle,T。(2006)。维护C。 elegans 。 WormBook 2(11)。
  8. Tomlinson,G.,Albuquerque,CA和Woods,RA(1985)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/3840091"target = "_blank"> amidantel(BAY d 8815)及其脱酰基衍生物(BAY d 9216)在秀丽隐杆线虫上的作用。 Eur J Pharmacol 113(2 ):255-262。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sant’Anna, V., de Souza, W. and Vommaro, R. C. (2017). Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model. Bio-protocol 7(2): e2113. DOI: 10.21769/BioProtoc.2113.
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