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The protoplasts assay constitutes a powerful tool that allows an easy uptake of active agents and a precise quantification of cell death induction in different populations. Our study showed that the basal level of cell death in our controls is low and stable throughout the length of our experiments (Danon et al., 2005; Pineau et al., 2013). In addition, the data obtained from the protoplast assay are applicable to intact seedlings, where it is possible to see differences in the intensity of necrotic lesions (Danon et al., 2006) even if those differences are not as easily and clearly quantifiable as with the protoplast assay.

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Protoplast Preparation and Determination of Cell Death
原生质体制备和细胞死亡的鉴定

植物科学 > 植物细胞生物学 > 细胞分离
作者: Antoine Danon
Antoine DanonAffiliation: UMR 8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Sorbonne Universités, UPMC Univ Paris 06, Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, Paris, France
For correspondence: antoine.danon@ibpc.fr
Bio-protocol author page: a1415
Vol 4, Iss 12, 6/20/2014, 4492 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1149

[Abstract] The protoplasts assay constitutes a powerful tool that allows an easy uptake of active agents and a precise quantification of cell death induction in different populations. Our study showed that the basal level of cell death in our controls is low and stable throughout the length of our experiments (Danon et al., 2005; Pineau et al., 2013). In addition, the data obtained from the protoplast assay are applicable to intact seedlings, where it is possible to see differences in the intensity of necrotic lesions (Danon et al., 2006) even if those differences are not as easily and clearly quantifiable as with the protoplast assay.
Keywords: Protoplast(原生质体), Arabidopsis thaliana(拟南芥), Cell death(细胞死亡), Evans blue(伊万斯蓝)

[Abstract]

Materials and Reagents

  1. Plant materials: Arabidopsis thaliana sterile seedlings grown in vitro from 1 to 3 weeks in the required conditions
  2. Gamborg B5 medium salt and vitamins (Duchefa Biochemie, catalog number: G0210 )
  3. 2-(N-Morpholino) ethanesulfonic acid (MES)
  4. Mannitol
  5. Digestion enzymes
    1. Cellulase (Duchefa Biochemie, catalog number: C8001 )
    2. Macerozyme (Duchefa Biochemie, catalog number: M8002 )
  6. Culture medium (see Recipes)
  7. Digestion medium (see Recipes)
  8. W5A medium (see Recipes)
  9. 21% sucrose (see Recipes)
  10. 1% Evans blue (see Recipes)

Equipment

  1. Light microscope
  2. An appropriate slide (e.g. Malassez slide)
  3. Centrifuge for 15 ml and 50 ml Falcon tubes
  4. Sterile pipette
  5. Filter (0.45 µm)
  6. Hemocytometer (VWR International, catalog number: 631-0975 )
  7. Cell dissociation sieve (100 µm) (Sigma-Aldrich, catalog number: CD1-1KT ) sieve mounted on a sterile 150 ml beaker
  8. Micropore tape (VWR International, catalog number: 115-8172 )

Procedure

  1. Protoplast extraction
    1.  Harvest whole young seedlings with sterile forceps and put them in a sterile Petri plate.
    2. Cover the leaves with the digestion solution (~15 ml for a 10 cm round plate).
    3. Close the plates with micropore tape and let digest at room temperature in the dark, overnight (~16 h).
    4. Separate the protoplasts from the seedlings by pipetting and releasing delicately the medium using a 3 ml sterile pipette.
    5. Filter the medium containing the protoplasts with a sieve of 100 µm, mounted on a sterile 150 ml beaker.
    6. Transfer the content of the beaker in a sterile 15 ml tube.
    7. Centrifuge 5 min at 180 x g at room temperature, eliminate the supernatant and resuspend the pellet in 4 ml of W5A medium.
    8. Very slowly deposit the protoplasts on 8 ml of 21% sucrose using a 3 ml sterile pipette (use a 50 ml tube if you have a lot of material, and a 15 ml tube otherwise).
    9. Centrifuge 13 min, 720 x g at room temperature.
    10. Carefully harvest the living protoplast at the surface of the sucrose solution (thin cushion, Figure 1) using a 3 ml sterile pipette and transfer them into a 15 ml sterile tube.


      Figure 1. Living and dead protoplasts after centrifugation on 21% sucrose

    11. Complete to 15 ml with W5A and calculate protoplast concentration on an aliquot using an appropriate slide (e.g. Malassez slide) and a light microscope (20x magnification).
    12. Centrifuge 5 min, at 180 x g at room temperature.
    13. Eliminate the supernatant, and resuspend the pellet in the appropriate volume of culture medium depending on the required concentration.

  2. Cell death determination
    1. Add 1 µl of 1% Evans blue to an aliquot of 25 µl of protoplast.
    2. Using light microscopy and an appropriate slide (e.g. Malassez slide), calculate the percentage of dead protoplasts that are stained with Evans Blue (blue protoplast/total protoplast, Figure 2).


      Figure 2. Control protoplast (left panel) and singlet oxygen-treated protoplast. Dark blue protoplasts are dead (Danon et al., 2005).

Recipes

  1. Culture medium (500 ml)
    Gamborg B5 medium salt and vitamins: 3.2 g
    0.4 M mannitol: 36.4 g
    0.4 M glucose: 36 g
    0.1% MES: 0.5 g
    Complete to 500 ml with distilled H2O
    Go to pH 5.7 with KOH
    Sterilize by autoclave and keep at 4 °C
  2. Digestion medium (500 ml)
    0.4 M mannitol: 36.4 g
    1% cellulase: 5 g
    0.25% macerozyme: 1.25 g
    8 mM CaCl2: 0.44 g
    0.1% MES: 0.5 g
    Complete to 500 ml with distilled H2O
    Homogenise until complete dissolution
    Go to pH 5.7 with KOH
    Sterilize by 0.45 µm filtration (it is easier to centrifuge before 5 min at 16,000 x g)
    Aliquot 10 ml and keep at -20 °C
  3. W5A medium (2,000 ml)
    5 mM glucose: 1.8 g
    154 mM Nacl: 18 g
    125 mM CaCl2: 27.8 g
    5 mM KCl: 0.76 g
    0.1% MES: 2 g
    Complete to 2,000 ml with distilled H2O
    Adjust to pH 5.7 with KOH
    Sterilize by autoclave and keep at RT
  4. 21% sucrose (100 ml)
    Sucrose: 21 g
    Complete to 100 ml with distilled H2O
    Sterilize by autoclave and keep at RT
  5. 1% Evans blue (100 ml)
    Dissolve 1 g Evans blue in 100 ml water

Acknowledgments

This work was supported by Sorbonne Universités, UPMC Univ Paris 06 and the Centre National de la Recherche Scientifique.

References

  1. Danon, A., Coll, N. S. and Apel, K. (2006). Cryptochrome-1-dependent execution of programmed cell death induced by singlet oxygen in Arabidopsis thaliana. Proc Natl Acad Sci U S A 103(45): 17036-17041.
  2. Danon, A., Miersch, O., Felix, G., Camp, R. G. and Apel, K. (2005). Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana. Plant J 41(1): 68-80.
  3. Pineau, B., Bourge, M., Marion, J., Mauve, C., Gilard, F., Maneta-Peyret, L., Moreau, P., Satiat-Jeunemaitre, B., Brown, S. C., De Paepe, R. and Danon, A. (2013). The importance of cardiolipin synthase for mitochondrial ultrastructure, respiratory function, plant development, and stress responses in Arabidopsis. Plant Cell 25(10): 4195-4208.

材料和试剂

  1. 植物材料:在所需条件下1至3周在体外生长的拟南芥无菌幼苗
  2. Gamborg B5中盐和维生素(Duchefa Biochemie,目录号:G0210)
  3. 2-(N-吗啉代)乙磺酸(MES)
  4. 甘露醇
  5. 消化酶
    1. 纤维素酶(Duchefa Biochemie,目录号:C8001)
    2. Macerozyme(Duchefa Biochemie,目录号:M8002)
  6. 培养基(见配方)
  7. 消化介质(参见配方)
  8. W5A介质(见配方)
  9. 21%蔗糖(见配方)
  10. 1%Evans蓝色(见配方)

设备

  1. 光学显微镜
  2. 适当的幻灯片(例如 Malassez幻灯片)
  3. 离心15 ml和50 ml Falcon管
  4. 无菌移液器
  5. 过滤器(0.45μm)
  6. 血球计(VWR International,目录号:631-0975)
  7. 安装在无菌150ml烧杯上的细胞离解筛(100μm)(Sigma-Aldrich,目录号:CD1-1KT)
  8. 微孔胶带(VWR International,目录号:115-8172)

程序

  1. 原生质体提取
    1.  用无菌镊子收获整个幼苗,并将它们放入无菌培养皿中
    2. 用消化溶液覆盖叶(〜15ml,对于10cm圆盘)。
    3. 用微孔胶带封闭板,在室温下在黑暗中消化过夜(约16小时)
    4. 通过移液和使用3ml无菌移液管轻轻释放培养基,从幼苗分离原生质体。
    5. 用安装在无菌150ml烧杯上的100μm筛网过滤含有原生质体的培养基
    6. 转移烧杯中的内容物在无菌15毫升管。
    7. 在室温下以180×g离心5分钟,除去 上清液并将沉淀重悬于4ml W5A培养基中。
    8. 非常缓慢地沉积原生质体在8毫升21%蔗糖使用3毫升   无菌移液管(使用50毫升管,如果你有很多材料,和a 否则为15ml管)
    9. 在室温下离心13分钟,720×g
    10. 小心收获活的原生质体在蔗糖的表面 溶液(薄垫,图1)使用3ml无菌移液管 将其转移到15ml无菌管中。


      图1.在21%蔗糖上离心后的活和死原生质体

    11. 用W5A完成至15ml,计算原生质体浓度 使用合适的载玻片(例如Malassez载玻片)和灯的等分试样 显微镜(放大20倍)。
    12. 在室温下以180×g离心5分钟。
    13. 消除上清液,并在适当的时候重悬沉淀   培养基的体积取决于所需的浓度。

  2. 细胞死亡测定
    1. 加入1微升1%伊文思蓝到25微升原生质体的等分试样
    2. 使用光学显微镜和适当的载玻片(例如Malassez载玻片)   计算被染色的死原生质体的百分比 伊文思蓝(蓝色原生质体/总原生质体,图2)

      图2。 控制原生质体(左图)和单线态氧处理的原生质体。深蓝色原生质体死亡(Danon等人,2005)。

食谱

  1. 培养基(500ml)
    Gamborg B5中盐和维生素:3.2g
    0.4M甘露醇:36.4g
    0.4M葡萄糖:36g
    0.1%MES:0.5g
    用蒸馏H 2 O 2完全达到500ml 用KOH调至pH 5.7,
    通过高压灭菌器灭菌并保持在4℃下
  2. 消化培养基(500ml)
    0.4M甘露醇:36.4g
    1%纤维素酶:5μg
    0.25%大分子酶:1.25g
    8mM CaCl 2:0.44g
    0.1%MES:0.5g
    用蒸馏的H 2 O完成至500ml 均匀至完全溶解
    用KOH调至pH 5.7,
    通过0.45μm过滤灭菌(在16,000×g下更容易在5分钟之前离心)
    等分10毫升,并保持在-20°C
  3. W5A培养基(2,000ml) 5mM葡萄糖:1.8g
    154mM Nacl:18g
    125mM CaCl 2:27.8g
    5mM KCl:0.76g
    0.1%MES:2g
    用蒸馏的H 2 O完成至2,000ml 用KOH调整到pH5.7 通过高压灭菌器灭菌并保持在室温下
  4. 21%蔗糖(100ml) 蔗糖:21g
    用蒸馏的H 2 O完成至100ml 通过高压灭菌器灭菌并保持在室温下
  5. 1%伊文思蓝(100ml) 将1g Evans蓝溶于100ml水中

致谢

这项工作得到Sorbonne大学,UPMC Univ巴黎06和中心国家研究中心的支持。

参考文献

  1. Danon,A.,Coll,N.S。和Apel,K。(2006)。 隐性铬1依赖性执行由单线态氧诱导的程序性细胞死亡拟南芥。 Proc Natl Acad Sci USA 103(45):17036-17041。
  2. Danon,A.,Miersch,O.,Felix,G.,Camp,R.G。和Apel,K。(2005)。 通过拟南芥中的单线态氧同时激活细胞死亡调节信号通路。 Plant J 41(1):68-80。
  3. Pineau,B.,Bourge,M.,Marion,J.,Mauve,C.,Gilard,F.,Maneta-Peyret,L.,Moreau,P.,Satiat-Jeunemaitre,B.,Brown,SC,De Paepe ,R.和Danon,A。(2013)。 心磷脂合成酶对线粒体超微结构,呼吸功能,植物发育和应激反应的重要性>拟南芥。植物细胞 25(10):4195-4208。
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How to cite this protocol: Danon, A. (2014). Protoplast Preparation and Determination of Cell Death. Bio-protocol 4(12): e1149. DOI: 10.21769/BioProtoc.1149; Full Text



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