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Cyst Detection in Toxoplasma gondii Infected Mice and Rats Brain
刚地弓形虫感染小鼠和大鼠脑部囊肿检测   

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Abstract

Toxoplasmosis caused by the intracellular parasite Toxoplasma gondii, is characterized by a life-long chronic infection. The parasite is an efficient neurotropic infectious agent that establishes its “safe” life by forming intracellular cysts in chronically infected animals and humans. This protocol describes the specific recipes and method to stain brain cysts from infected mice and rats for further quantification using epifluorescence microscopy. This method provides the possibility to scan the entire brain and thus to numerate all cysts.

Keywords: Toxoplasma gondii(弓形虫), Cysts(囊肿), Epifluorescence microscopy(荧光显微技术), Dolichos(扁豆), Quantification(量化)

Materials and Reagents

  1. Human foreskin fibroblasts (HFFs) cells culture (ATTC, catalog number: SCRC-1042 )
  2. Dulbecco's modified Eagle medium (DMEM) (Life Technologies, catalog number: 41966-029/052 )
  3. Fetal bovine serum (FBS) (Eurobio®, catalog number: CVFSVF0001 )
  4. Penicillin/streptomycin (PAN Biotech GmbH, catalog number: P0607-100 )
  5. L-Glutamine (200 mM) (Life Technologies, catalog number: 25030-024 )
  6. Dulbecco's phosphate-buffered saline (DPBS) (Life Technologies, catalog number: 14190-094/069 )
  7. Proteinase K (molecular biology grade) (Bio-Rad Laboratories, catalog number: P8107S , Lot: 0051310)
  8. TRIS (Euromedex, catalog number: 26-128-3094-B )
  9. EDTA (Euromedex, catalog number: E013 )
  10. SDS (Euromedex, catalog number: EU0660 )
  11. NaCl (Euromedex, catalog number: 1112-A )
  12. HCl (37% ACS reagent) (Sigma-Aldrich, catalog number: 258148-2.5ML )
  13. Phenylmethylsulfonylfluoride (PMSF) (Euromedex, catalog number: 1111-C )
  14. Hoescht 33258 (Life Technologies, Molecular Probes®, catalog number: H-3569 )
  15. Formaldehyde methanol free (Polysciences, catalog number: 0 4018 )
  16. Dolichos biflorus lectin coupled to fluorescein isothiocyanate (FITC) (Clinisciences, catalog number: FL-1031 )
  17. Complete DMEM medium (see Recipes)
  18. Phenylmethylsulfonylfluoride (PMSF) solution (see Recipes)
  19. Lysis buffer (see Recipes)

Equipment

  1. 37 °C/5% CO2 cell culture incubator
  2. 6-well plates
  3. Scissors and forceps
  4. Glass homogenizers (Potter-Elvehjem PTFE, 15 ml) (Dutscher, catalog numbers: 057009 and 057021 )
  5. 15 ml polystyrene tubes
  6. Epifluorescence microscope

Procedure

  1. Preparation of Human Foreskin Fibroblasts cell culture in 6-well plates
    Plate HFFs cells in complete DMEM medium and culture them for 4 days at 37 °C in presence of CO2. HFFs have to be at 100% confluence in each well (8 x 105 cells) before staining.

  2. Staining of HFFs
    1. Wash twice the cells culture with 1 ml 1x DPBS.
    2. Fix cells with 1 ml formaldehyde diluted at 2.5% in DPBS 1x for 20 min at 4 °C.
    3. Incubate 20 min, in the dark and at RT, the cells with 1 ml of Hoescht diluted 1/50,000 in 1x DPBS.
    4. Wash twice the cells culture with 1 ml 1x DPBS.
    5. Add 1 ml 1x DPBS.
    6. Stored at 4 °C until addition of the stained homogenized brain.
      Note: The stained cells are stored at 4 °C for a maximum of 24 h.

  3. Isolation of brains from infected mice or rats
    1. Anesthesize animal with isofluoran and euthanize it by cervical dislocation.
    2. Soak the head with 70% (v/v) ethanol.
    3. Cut the skin at the base of skull and remove it as much as possible.
    4. Cut with scissors the dorsal and lateral part of the skull and take the top off.
    5. Collect the brain tissue in 5 ml DPBS 1x solution.

  4. Brains homogenization
    Put each mouse brain in 2 ml DPBS 1x into a glass homogenizer, homogenize at RT and adjust final volume to 4 ml. For rat brain, homogenize in 4 ml and adjust to 8 ml final.
    Note: Brains can be stored at 4 °C until the staining for no more than 2 days.

  5. Brains staining
    1. Prepare 5x lysis buffer and proteinase K at 8 U/ml (stock at 800 U/ml, 1/100 dilution in 5x lysis buffer just before use).
    2. For mice, take 1 ml of homogenized brain (¼ brain), add 398 µl of 5x lysis buffer, 2 µl of proteinase K (see step E1, so final concentration at 0.008 U/ml) and 600 µl of 1x DPBS (final volume of 2 ml).
      For rats, take 2 ml of homogenized brain (¼ brain), add 995 µl of 5x lysis buffer, 5 µl of proteinase K (see step E1, so final concentration at 0.008 U/ml) and 2 ml of 1x DPBS (final volume of 5 ml).
    3. Incubate at 56 °C for 15 min, homogenize every 5 min by vortexing.
    4. Stop the proteinase K activity by adding PMSF to a final concentration of 2 mM (stock at 200 mM), homogenize and incubate at RT for 5 min.
    5. Centrifuge the sample for 15 min at 1,250 x g (RT).
    6. Gently eliminate the supernatant by pipetting.
    7. Resuspend in 1 ml of Dolichos biflorus lectin diluted at 1/250 in DPBS 1x (996 µl 1x DPBS + 4 µl of Dolichos biflorus lectin).
    8. Incubate 30 min at RT in a mechanical wheel placed in the dark.
    9. Add 3 ml DPBS 1x and homogenize to wash the sample.
    10. Centrifuge 15 min at 1,250 x g (RT).
    11. Gently eliminate the supernatant by pipetting.
    12. Resuspend each sample in 1 ml in DPBS 1x and transfer onto Hoescht-stained HFFs (see part A and B of the procedure) by pipetting (1 ml/well).
      Note: It is important to transfer the homogenized brain pellet onto HFF cells to facilitate microscope focus when there are no or very few cysts.

  6. Count the cysts using epifluorescence microscopy (Representative data)
    Note: Samples can be stored at 4 °C until microscope observation for no more than 10 days. To know the cysts quantity in entire brain, multiply the counted number by 4.

Representative data



Figure 1. Representative cysts staining from uninfected and infected CBA mice brain with Toxoplasma gondii parasites. Two months after i.p. infection of CBA mice with 1,000 ΔKu80-ΔHXGPRT (PRU) tachyzoïtes, brains were collected, homogenized in 4 ml of PBS and ¼ of each brain suspension was used for the labeling of cyst walls with the D. biflorus-FITC lectin and analyzed by high content screening microscopy (Scan^R Olympus). A) Photo (4x objective) of a CBA infected mouse brain. Three stained cysts are present in this field. B) Uninfected CBA mouse brain (4x objective) no cysts are visible but only brain debris. C) Representative panel of stained cysts obtained from infected CBA mouse and detected by (4x objective) microscopy. Cysts are zoomed to detect potential false positive as in A6.

Recipes

  1. Complete DMEM medium
    10% (v/v) FBS
    0.5% (v/v) penicillin/streptomycin
    2 mM glutamine
  2. Phenylmethylsulfonylfluoride (PMSF) solution (stock concentration of 200 mM)
    0.35 g of PMSF powder to dissolve in 10 ml of isopropanol
    Stored at -20 °C in 1 ml aliquots
    Note: PMSF will take some time to dissolve.
  3. Lysis buffer (500 ml)
    10 M TRIS: 2.5 ml of 2 M TRIS at pH 8.0 (60 g in 250 ml H2O, adjust pH)
    1 mM EDTA: 2 ml of 250 mM EDTA at pH 8.0 (23.27 g in 250 ml H2O, adjust pH)
    0.2% (w/v) SDS: 5 ml of 20% (w/v) SDS
    100 mM NaCl (1.165 g)
    Up to 500 ml H2O
    Note: The 8.0 pH is adjusted using HCI 3 M (HCl 3 M: 25 ml HCl 37% added to 75 ml of H2O).

Acknowledgments

This work was supported by the ANR grants Nu 05-MIIM-020-02 and Nu 07-MIME-013-01, the Lyonbiopole competitiveness cluster and the French Parasitology consortium ParaFrap (ANR-11-LABX0024).

References

  1. Aldebert, D., Hypolite, M., Cavailles, P., Touquet, B., Flori, P., Loeuillet, C. and Cesbron-Delauw, M. F. (2011). Development of high-throughput methods to quantify cysts of Toxoplasma gondii. Cytometry A 79(11): 952-958.
  2. Cavailles, P., Flori, P., Papapietro, O., Bisanz, C., Lagrange, D., Pilloux, L., Massera, C., Cristinelli, S., Jublot, D., Bastien, O., Loeuillet, C., Aldebert, D., Touquet, B., Fournie, G. J. and Cesbron-Delauw, M. F. (2014). A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage. PLoS Pathog 10(4): e1004005.

简介

由胞内寄生虫弓形虫引起的弓形虫病的特征在于终生的慢性感染。 寄生虫是一种有效的亲神经感染剂,通过在慢性感染的动物和人中形成细胞内囊肿来建立其"安全"的生命。 该协议描述了从感染的小鼠和大鼠染色脑囊肿的具体配方和方法,以进一步使用落射荧光显微镜定量。 这种方法提供扫描整个大脑,从而计算所有囊肿的可能性。

关键字:弓形虫, 囊肿, 荧光显微技术, 扁豆, 量化

材料和试剂

  1. 人包皮成纤维细胞(HFF)细胞培养物(ATTC,目录号:SCRC-1042)
  2. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,目录号:41966-029/052)
  3. 胎牛血清(FBS)(Eurobio ,目录号:CVFSVF0001)
  4. 青霉素/链霉素(PAN Biotech GmbH,目录号:P0607-100)
  5. L-谷氨酰胺(200mM)(Life Technologies,目录号:25030-024)
  6. Dulbecco's磷酸盐缓冲盐水(DPBS)(Life Technologies,目录号:14190-094/069)
  7. 蛋白酶K(分子生物学级)(Bio-Rad Laboratories,目录号:P8107S,批号:0051310)
  8. TRIS(Euromedex,目录号:26-128-3094-B)
  9. EDTA(Euromedex,目录号:E013)
  10. SDS(Euromedex,目录号:EU0660)
  11. NaCl(Euromedex,目录号:1112-A)
  12. HCl(37%ACS试剂)(Sigma-Aldrich,目录号:258148-2.5ML)
  13. 苯基甲基磺酰氟(PMSF)(Euromedex,目录号:1111-C)
  14. Hoescht 33258(Life Technologies,Molecular Probes ,目录号:H-3569)
  15. 不含甲醛甲醇(Polysciences,目录号:04018)
  16. 偶联异硫氰酸荧光素(FITC)(Clinisciences,目录号:FL-1031)的双歧杆菌凝集素
  17. 完成DMEM培养基(参见配方)
  18. 苯基甲基磺酰氟(PMSF)溶液(参见配方)
  19. 裂解缓冲液(见配方)

设备

  1. 37℃/5%CO 2细胞培养孵化器
  2. 6孔板
  3. 剪刀和镊子
  4. 玻璃均化器(Potter-Elvehjem PTFE,15ml)(Dutscher,目录号:057009和057021)
  5. 15 ml聚苯乙烯管
  6. 荧光显微镜

程序

  1. 制备人类包皮成纤维细胞在6孔板中培养 将板HFFs细胞在完全DMEM培养基中并在CO 2存在下在37℃下培养4天。 在染色之前,HFF在每个孔(8×10 5个细胞)中必须达到100%汇合。

  2. HFF的染色
    1. 用1ml 1x DPBS洗涤细胞培养物两次。
    2. 修复细胞用1ml甲醛在2.5%在DPBS 1x稀释在4℃下20分钟。
    3. 孵育20分钟,在黑暗中和在室温下,细胞用1ml的Hoescht稀释1/50,000在1x DPBS中。
    4. 用1ml 1x DPBS洗涤细胞培养物两次。
    5. 加入1ml 1x DPBS。
    6. 在4℃保存,直到加入染色的均质脑。
      注意:染色的细胞在4℃下储存最多24小时。

  3. 从受感染的小鼠或大鼠中分离大脑
    1. 麻醉动物与异氟醚和安乐死通过颈脱位。
    2. 用70%(v/v)乙醇浸泡头部
    3. 切开颅骨底部的皮肤,尽可能多地除去
    4. 用剪刀切开头骨的背部和侧面部分,取下顶部
    5. 收集脑组织在5ml DPBS 1x溶液。

  4. 大脑均匀化
    将每只小鼠脑中的2毫升DPBS 1x放入玻璃匀浆器,匀浆在室温和调整终体积为4毫升。 对于大鼠脑,在4ml中匀浆,并调整至8ml最终。
    注意:大脑可以储存在4°C,直到染色不超过2天。

  5. 脑炎染色
    1. 准备5 x裂解缓冲液和蛋白酶K 8 U/ml(库存800 U/ml,1/100稀释在5x裂解缓冲液在使用前)。
    2. 对于小鼠,取1ml均质脑(1/4脑),加入398μl5x   裂解缓冲液,2μl蛋白酶K(参见步骤E1,因此最后 浓度为0.008U/ml)和600μl1×DPBS(终体积为2μl) ml) 对于大鼠,取2ml均质脑(1/4脑),加入995μl 的5×裂解缓冲液,5μl蛋白酶K(参见步骤E1,因此最终 浓度为0.008U/ml)和2ml 1×DPBS(终体积为5μl) ml)。
    3. 在56℃孵育15分钟,通过涡旋匀化每5分钟
    4. 通过添加PMSF到最终,停止蛋白酶K活动 浓度为2mM(200mM的原液),匀浆并在室温下孵育 5分钟。
    5. 在1250×g(RT)下离心样品15分钟。
    6. 轻轻吸取上清液。
    7. 重悬于1ml用1/250英寸稀释的Dolichos biflorus凝集素 DPBS 1×(996μl1×DPBS +4μl的双岐菌凝集素)。
    8. 在室温下放置在黑暗中的机械轮中孵育30分钟
    9. 加入3ml DPBS 1x并均质以洗涤样品
    10. 在1250×g(RT)下离心15分钟。
    11. 轻轻吸取上清液。
    12. 重悬每个样品在1毫升在DPBS 1x和转移到 Hoescht染色的HFFs(参见程序的A部分和B部分)   ml /孔) 注意:重要的是转移均质脑 沉淀到HFF细胞上,以便在没有时显微镜聚焦 或非常少的囊肿。

  6. 使用落射荧光显微镜(代表数据)计数囊肿
    注意:样品可以在4℃下储存,直到显微镜观察不超过10天。 要知道整个大脑的囊肿数量,将计数值乘以4。

代表数据



图1.来自未感染和感染的CBA小鼠的代表性囊肿,具有弓形虫寄生虫的脑。 i.p.后两个月。用1000个ΔKu80-ΔHXGPRT(PRU)速殖子感染CBA小鼠,收集脑,在4ml PBS中匀浆,并将1/4脑的每种悬浮液用于用D标记囊肿壁。并通过高含量筛选显微镜(Scan ^ R Olympus)进行分析。 A)CBA感染的小鼠脑的照片(4倍目标)。这个领域中存在三个染色的囊肿。 B)未感染的CBA小鼠脑(4倍目的),没有可见的囊肿,只有脑碎片。 C)从感染的CBA小鼠获得的染色囊肿的代表性组并通过(4x物镜)显微镜检测。囊肿被放大以检测潜在的假阳性,如在A6中。

食谱

  1. 完成DMEM培养基
    10%(v/v)FBS
    0.5%(v/v)青霉素/链霉素 2mM谷氨酰胺
  2. 苯基甲基磺酰氟(PMSF)溶液(储存浓度为200mM) 将0.35g PMSF粉末溶于10ml异丙醇中 储存于-20℃,1ml等份
    注意:PMSF需要一些时间来解散。
  3. 裂解缓冲液(500ml) 10μMTRIS:2.5ml pH 8.0的2M TRIS(在250ml H 2 O中60g,调节pH)
    1mM EDTA:2ml pH 8.0的250mM EDTA(在250ml H 2 O中23.27g,调节pH)
    0.2%(w/v)SDS:5ml 20%(w/v)SDS 100mM NaCl(1.165g) 高达500ml H 2 O v/v 注意:使用HCl 3 M(HCl 3 M:25ml HCl 37%加入到75ml H 2 SO 4中, em> O)。

致谢

这项工作由ANR授予Nu 05-MIIM-020-02和Nu 07-MIME-013-01,Lyonbiopole竞争力集群和法国寄生物学协会ParaFrap(ANR-11-LABX0024)支持。

参考文献

  1. Aldebert,D.,Hypolite,M.,Cavailles,P.,Touquet,B.,Flori,P.,Loeuillet,C.and Cesbron-Delauw,M.F。 开发高通量方法以量化弓形虫的囊肿。 Cytometry A 79(11):952-958。
  2. Cavailles,P.,Flori,P.,Papapietro,O.,Bisanz,C.,Lagrange,D.,Pilloux,L.,Massera,C.,Cristinelli,S.,Jublot,D.,Bastien, Loeuillet,C.,Aldebert,D.,Touquet,B.,Fournie,GJ和Cesbron-Delauw,MF(2014)。 高度保守的Toxo1单倍型直接抵抗弓形体病和其相关的胱天蛋白酶-1依赖性杀死寄生虫和宿主巨噬细胞。 PLoS Pathog 10(4):e1004005。
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Copyright: © 2015 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. Bellini, V., Loeuillet, C., Massera, C., Cesbron-Delauw, M. and Cavaillès, P. (2015). Cyst Detection in Toxoplasma gondii Infected Mice and Rats Brain. Bio-protocol 5(7): e1439. DOI: 10.21769/BioProtoc.1439.
  2. Cavailles, P., Flori, P., Papapietro, O., Bisanz, C., Lagrange, D., Pilloux, L., Massera, C., Cristinelli, S., Jublot, D., Bastien, O., Loeuillet, C., Aldebert, D., Touquet, B., Fournie, G. J. and Cesbron-Delauw, M. F. (2014). A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage. PLoS Pathog 10(4): e1004005.
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