搜索

Quantification of Membrane Damage/Cell Death Using Evan’s Blue Staining Technique
采用伊文思蓝染色技术定量膜损伤/细胞死亡   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Membrane damage is a hallmark of both biotic and abiotic stress responses. The membrane determines the ability of a cell to sustain altered environmental conditions and hence can be used as a biomarker to assess stress-induced cell damage or death. We present an easy, quick, cost-effective, staining and spectrophotometric method to assess membrane stability of plant cells. In this method, Evan’s blue, an azo dye, is used to assay for cell viability. More specifically, Evan’s blue dye can penetrate through ruptured or destabilized membranes and stain cells. Thus, when plant cells are subjected to stress that compromises membrane integrity, the number of cells that are permeated by Evan’s blue dye will be increased compared to control cells that are not stressed. In contrast, live, healthy cells that are capable of maintaining membrane integrity do not take up Evan’s blue dye. Cells that have taken up Evan’s blue dye will have an accumulation of a blue protoplasmic stain and these stained cells can be qualitatively documented under bright field microscopy with or without the use of a camera. Furthermore, the dye can be extracted from cells that are stained by Evan’s blue dye and can be quantified spectrophotometrically. Using this analysis, the accumulation of dye in positively-stained cells correlates with the extent of cell membrane damage and thus the amount of cells that are stained with Evan’s blue dye under various conditions can be used as an indicator of cellular stress.

Keywords: Evan’s blue(伊文思蓝), Membrane damage(膜损伤), Cell death(细胞死亡)

Background

Plants are sessile organisms that are exposed to a diverse array of stress factors. The membrane is made up of lipids and glycoproteins and act as a physical, protective barrier. The fluidity of the cell membrane is altered when the cell is exposed to stress such as heat. Oxidative stress can damage cell membranes. The reactive oxygen species (ROS) associated with oxidative stress can act on membrane lipids to decrease membrane stability. An established protocol to assess membrane stability, known as Sullivan’s method, quantifies the extent of electrolyte leakage from the membrane (Sullivan and Ross, 1979). This method is time-consuming, tedious and involves several steps. Additionally, since this method usually requires exposure of the tissue to high temperature (Initial electrolyte leakage and final electrolyte leakage after boiling at high temperature), this method cannot be used to assess the instantaneous damage to membranes in plants exposed to stress. We adapted a reliable Evan’s blue staining technique that has been used by many researchers to assess cell death or membrane damage (Smith et al., 1982; Oprisko et al., 1990; Vemanna et al., 2017) for instantly monitoring stress. Evan’s blue is an acidic, non-permitting exclusion dye which stains dead or damaged cells. The dye does not enter live cells with stable membranes (Gaff and Okong’O-Ogala, 1971). One advantage of this method is that it does not subject the tissue to high temperature. Though microscopic visualization is effective, large sample sizes make this type of analysis too time consuming (Baker and Mock, 1994). We have altered our method to analyze spectrophotometrically. Evan’s blue stain can be extracted from intact cells and analyze by spectrophotometer. Our method is highly reproducible and it can be adapted to large scale phenotyping of genotypes. The other membrane penetrating dye phenosafranin can also be used but some difficulties have been reported that it will not stain the cells without nuclei (ghost cell) and also the uptake is affected by pH (Baker and Mock, 1994).

Materials and Reagents

  1. Eppendorf tubes
  2. Petri plates of 10 cm diameter
  3. 96-well plates or ELISA plate (Thermo Fisher Scientifc, Thermo ScientifcTM, catalog number: 442404 ) or cuvette (Sigma-Aldrich, catalog number: Z276758 )
  4. Leaf or root tissue collected during stress period (approximately 10 discs or 250 mg)
  5. Distilled water
  6. Evan’s blue (Sigma-Aldrich, catalog number: E2129 )
  7. Sodium chloride (NaCl)
  8. Potassium nitrate (KNO3)
  9. Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O
  10. Ammonium dihydrogen phosphate (NH4H2PO4)
  11. Magnesium sulfate heptahydrate (MgSO4·7H2O)
  12. Potassium chloride (KCl)
  13. Boric acid (H3BO3)
  14. Manganese sulfate (MnSO4·H2O)
  15. Zinc sulfate heptahydrate (ZnSO4·7H2O)
  16. Copper(II) sulfate pentahydrate (CuSO4·5H2O)
  17. Molybdic acid (H2MoO4)
  18. Na·Fe·DTPA
  19. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  20. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  21. Hydrochloric acid (HCl)
  22. Evan’s blue staining solution (see Recipe 1)
  23. Hoagland solution (see Recipe 2)
  24. 0.1 M CaCl2 of pH 5.6 (see Recipe 3)
  25. 1% SDS (see Recipe 4)

Equipment

  1. Pipettes
  2. (Optional) Pestle and mortar
  3. Tissue lyser (Tissue lyser II) (QIAGEN, catalog number: 69982 )
  4. Centrifuge (Thermo Fisher Scientific, model: SorvallTM ST16 , catalog number: 75004240)
  5. Light microscope (MAGNUS ANALYTICS, model: MLX )
  6. pH meter (Systronics, model: µController Based pH system 361, catalog number: 361 )
  7. Spectrophotometer/ELISA plate reader (Molecular Devices, model: SpectraMax Plus 384 )
  8. Orbital shaker (Shalom Instruments, model: SLM-GR-100 )

Procedure

  1. Prepare Evan’s blue solution (see Recipe 1)
  2. Stain tissue with Evan’s blue to assess extent of membrane stability
    1. Grow the plants under normal conditions.
    2. Impose the stress treatment to be determined for specific growth stages of plants and collect the tissue (leaf or root).
      For example, we imposed the stress to leaves that are collected from 30-day-old plants of groundnut. We perform the leaf disc assay with small leaf discs of 1 cm diameter. We transfer ten leaf discs to a Petri plate and then impose methyl viologen (MV)-induced oxidative stress treatment. Methyl viologen at 5 µM is exposed to one set (stress) and another set is kept in pure water (control) and these leaf discs exposed to high light conditions (1,000 µmol m-2 sec-1) in room temperature (RT) for three hours. These leaf discs, were then stained with Evan’s blue stain (Figure 1A).
    3. In another set of experiments, NaCl (150 mM in Hoagland solution [see Recipe 2]) stress is imposed to one set of rice seedlings (stress) and the other set was kept only in Hoagland’s solution (control) in a hydroponic experiment (Lopez and Satti, 1996). Three days after stress imposition collect the roots from control and stressed plants for Evan’s blue staining and membrane stability determination (Figure 1B). This method is highly reproducible with ~95% accuracy
    4. Transfer the collected leaf and root tissues to 2 ml Eppendorf tubes and add 2 ml of Evan’s blue solution to each tube.
      Note: Adjust the volume to ensure that tissues are immersed in the solution.


      Figure 1. Determination of membrane stability using Evan’s blue staining. Microscopic detection of Evan’s blue stain in (A) methyl viologen (MV) 5 µM treated leaf discs and (B) 150 mM NaCl treated roots. Scale bar indicates 25 µm. The groundnut leaf discs from 30-day-old plants were incubated in MV for 3 h at high light intensity of 1,000 µmol m-2 sec-1 and subsequently the leaf discs were stained with Evan’s blue dye. For NaCl stress the rice seedlings of 30-days old were exposed to 150 mM NaCl for 3 days and the roots were stained with Evan’s blue dye to assess membrane integrity.

    5. Shake the tubes in an orbital shaker at 50 oscillations/min for 20 min.
      Note: Shaking is just to ensure that all of the tissue is in contact with the Evan’s blue dye solution.
    6. Wash the roots and leaves thoroughly with distilled water thrice or until unbound dye washes out from surface.
      Note: (Optional) Root or leaf tissue can also be washed with 0.1 M CaCl2 of pH 5.6 (see Recipe 3).
    7. Observe the stained leaf or root under the normal, transmitted light microscope. Examine the leaves or roots and take photograph of images under a brightfield microscope. Furthermore, the Evan’s blue stained images can be quantified using imageJ software to assess the viability of cells.
      Note: If the sample size is large, it’s preferable to follow only spectrophotometric quantification and appropriate statistics can be applied to arrive at significance levels.
  3. Quantification Evan’s blue stain taken up due to membrane destabilization
    1. To extract the Evan’s blue dye from the roots and leaves, use 1 ml of 1% SDS (see Recipe 4) cell lysis buffer which breaks up membrane structures.
    2. Grind 100 mg of tissue using a tissue lyser with a frequency of 15 strokes per second for 10 min, if the sample number is large. If only a few samples are needed, they can be ground in a pestle and mortar. After grinding, transfer the extract to 2 ml Eppendorf tubes.
    3. Centrifuge the extract at 7,168 x g for 5 min at RT to elute the dye in to the solution and to remove the debris.
    4. Transfer an aliquot of supernatant to new tubes or directly transfer 250 µl of supernatant to a 96-well microtiter plate.
      Note: Since Evan’s blue dye will not oxidize, it can be stored at RT for half an hour to measure large samples.
    5. Measure the optical density at 600 nm spectrophotometrically using a cuvette or a plate reader by taking 1% SDS as blank.
    6. Concentration of Evan’s blue can be estimated by referring to a standard curve (Figure 2).
    7. Plot the graph of concentration of Evan’s blue on y-axis and plant number or stress treatment on x-axis (Figure 3A and 3B).


      Figure 2. Standard graph for Evan’s blue staining. Standard graph is prepared using 1 µg ml-1 of Evan’s blue stock solution. Different concentrations of Evan’s blue were diluted and absorbance was recorded at 600 nm.


      Figure 3. Quantification of membrane stability using the spectrophotometric method. Histogram showing the extent of Evan’s blue dye taken up by the (A) leaf tissue and (B) root tissue as a reflection of membrane damage. The extent of dye taken up by the cells was quantified by grinding the tissue in 1% sodium dodecyl sulfate (SDS) and measuring the absorbance at 600 nm and calculated using standard graph. Error bars indicate the minimum of three replicates data. * indicate the least significant difference (LSD) at P < 0.05 analyzed using GenStat statistical tool.

  4. Preparation of standard curve using Evan’s blue:
    Dissolve 1 µg of Evan’s blue in 1 ml of distilled water to get 1 µg ml-1 stock solution. From stock solution make five concentrations by making up the volume to 1 ml. The absorbance was measured at 600 nm in spectrophotometer (Figure 2).

Data analysis

A minimum of three biological replicates were used to quantify the uptake of Evan’s blue dye and the data were analyzed using the GenStat program (Marcos et al., 2013). Significance level was tested using analysis of variance (ANOVA) depending on the number of genotypes and treatment levels.

Recipes

  1. Evan’s blue staining solution
    Dissolve 0.25 g of Evan’s blue dye in 100 ml of 0.1 M CaCl2 solution at pH 5.6 and mix well until it is dissolved
    Note: Evan’s blue solution should be freshly prepared each time.
  2. Hoagland solution
    Composition of Hoagland solution used in this experiment is given in Table 1.

    Table 1. Composition of Hoagland solution

    *Note: This should be avoided in salinity stress treatment imposition.

  3. 0.1 M CaCl2 of pH 5.6
    To prepare 0.1 M CaCl2, dissolve 1.10 g of CaCl2 (Molecular Weight = 110.989) in distilled water (1 L) and adjust pH to 5.6 using 0.2 N HCl. CaCl2 solution can be stored for a month in RT
  4. 1% SDS
    To prepare 1% SDS, add 1 g of sodium dodecyl sulfate to 80 ml distilled water and make up the volume to 100 ml. SDS should be prepared freshly before the use. Optionally it can be stored for a month in RT

Acknowledgments

Indian Council of Agricultural Research–Niche Area of Excellence program (F. No. 10-(6)/2005 EPD) and (F. No. 10 (15) 2012 EPD). MUK acknowledges the support for platinum jubilee fellowship from NASI, India.

References

  1. Baker, C. J. and Mock, N. M. (1994). An improved method for monitoring cell death in cell suspension and leaf disc assay using Evan’s blue. Plant Cell Tissue Organ Cult 39(1): 7-12.
  2. Gaff, D. F. and Okong'O-Ogola, O. (1971). The use of non-permeating pigments for testing the survival of cells. J Exp Bot 22: 756-758.
  3. Lopez, M. V. and Satti, S. M. E. (1996). Calcium and potassium-enhanced growth and yield of tomato under sodium chloride stress. Plant Science 114: 19-27.
  4. Marcos, M., Jean-Marcel, R. and Fred, A. V. (2013). The statistical analysis of multi environment data: modelling genotype-by-environment interaction and its genetic basis. Front Physiol 4: 44.
  5. Oprisko, M. J., Green, R. L., Beard, J. B. and Gates, C. E. (1990). Vital staining of root hairs in 12 warm-season perennial grasses. Crop Sci 30: 947-950.
  6. Vemanna, R. S., Babitha, K. C., Solanki, J. K., Amarnatha Reddy, V., Sarangi, S. K. and Udayakumar, M. (2017). Aldo-keto reductase-1 (AKR1) protect cellular enzymes from salt stress by detoxifying reactive cytotoxic compounds. Plant Physiol Biochem 113: 177-186.
  7. Smith, B. A., Reider, M. L. and Fletcher, J. S. (1982). Relationship between vital staining and subculture growth during the senescence of plant tissue cultures. Plant Physiol 70: 1228-1230.
  8. Sullivan, C. Y. and Ross, W. M. (1979). Selecting for drought and heat resistance in grain sorghum. In: Mussell, H. and Staples, R.C. (Eds.). Stress Physiology in Crop Plants. John Wiley and Sons pp: 263-281.

简介

膜损伤是生物和非生物胁迫反应的标志。膜决定细胞维持改变的环境条件的能力,因此可以用作生物标志物来评估应激诱导的细胞损伤或死亡。我们提出了一种简单,快速,经济有效的染色和分光光度法来评估植物细胞的膜稳定性。在这种方法中,Evan的蓝色偶氮染料被用于测定细胞活力。更具体地说,Evan的蓝色染料可以穿透破裂或不稳定的膜并染色细胞。因此,当植物细胞受到影响膜完整性的应激时,与不受应激的对照细胞相比,Evan蓝染料渗透的细胞数将增加。相比之下,能够保持膜完整性的活的健康细胞不会吸收Evan的蓝色染料。摄取Evan蓝色染料的细胞会积累蓝色原生质染料,这些染色的细胞可以在有或没有使用相机的明视野显微镜下进行定性记录。此外,染料可以从被Evan的蓝色染料染色的细胞中提取,并且可以通过分光光度法进行定量。使用这种分析,染色阳性染色细胞中染料的积累与细胞膜损伤的程度相关,因此在各种条件下用Evan蓝色染料染色的细胞数量可用作细胞应激的指标。
【背景】植物是暴露于各种紧张因素的固着生物。膜由脂质和糖蛋白组成,作为物理保护屏障。当细胞暴露于诸如热的应力时,细胞膜的流动性被改变。氧化应激会破坏细胞膜。与氧化应激相关的活性氧(ROS)可以作用于膜脂质以降低膜的稳定性。评估膜稳定性的一个已建立的方案,称为沙利文方法,量化了膜中电解质渗漏的程度(Sullivan和Ross,1979)。这种方法是耗时的,繁琐的,涉及几个步骤。另外,由于该方法通常需要将组织暴露于高温(初始电解质泄漏和在高温沸腾后的最终电解质泄漏),所以该方法不能用于评估暴露于应力的植物中对膜的瞬时损伤。我们调整了一种可靠的Evan蓝染色技术,被许多研究人员用于评估细胞死亡或膜损伤(Smith等,1982; Oprisko等,1990; Vemanna等,2017),用于立即监测应激。 Evan的蓝色是一种酸性,不允许排除染料,污染死亡或损坏的细胞。染料不能进入具有稳定膜的活细胞(Gaff和Okong'O-Ogala,1971)。该方法的一个优点是它不会使组织受到高温。虽然微观可视化是有效的,但大样本量使得这种类型的分析太费时(Baker和Mock,1994)。我们改变了我们的分光光度法分析方法。 Evan的蓝色染色可以从完整的细胞中提取出来,并通过分光光度计进行分析。我们的方法是高度可重复的,它可以适应于大规模表型的基因型。也可以使用另一种膜穿透染料phenosafranin,但有一些困难已被报道,它不会染色细胞没有细胞核(鬼细胞),并且摄取受pH影响(Baker和Mock,1994)。

关键字:伊文思蓝, 膜损伤, 细胞死亡

材料和试剂

  1. Eppendorf管
  2. 10厘米直径的Petri板
  3. 96孔板或ELISA板(Thermo Fisher Scientifc,Thermo Scientifc TM,目录号:442404)或比色皿(Sigma-Aldrich,目录号:Z276758)
  4. 在应激期间收集叶或根组织(约10片或250毫克)
  5. 蒸馏水
  6. 埃文蓝(Sigma-Aldrich,目录号:E2129)
  7. 氯化钠(NaCl)
  8. 硝酸钾(KNO 3 )
  9. 硝酸钙四水合物,Ca(NO 3 3)2·4H 2 O - / -
  10. 磷酸二氢铵(NH 4 H 2 PO 4)&lt;
  11. 七水硫酸镁(MgSO 4·7H 2 O)
  12. 氯化钾(KCl)
  13. 硼酸(H 3 3 BO 3)
  14. 硫酸锰(MnSO 4·H 2 O)
  15. 硫酸锌七水合物(ZnSO 4·7H 2 O)
  16. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)
  17. 钼酸(H 2 O 3 MoO 4)
  18. Na·Fe·DTPA
  19. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  20. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  21. 盐酸(HCl)
  22. Evan的蓝色染色溶液(参见配方1)
  23. Hoagland解决方案(请参阅配方2)
  24. pH值为5.6的0.1M CaCl 2(参见方法3)
  25. 1%SDS(参见配方4)

设备

  1. 移液器
  2. (可选)杵和砂浆
  3. 组织裂解器(组织溶解酶II)(QIAGEN,目录号:69982)
  4. 离心机(Thermo Fisher Scientific,型号:Sorvall TM ST16,目录号:75004240)
  5. 光学显微镜(MAGNUS ANALYTICS,型号:MLX)
  6. pH计(Systronics,型号:μControllerBased pH系统361,目录号:361)
  7. 分光光度计/ ELISA读板仪(Molecular Devices,型号:SpectraMax Plus 384)
  8. 轨道摇床(Shalom Instruments,型号:SLM-GR-100)

程序

  1. 准备Evan的蓝色解决方案(请参阅配方1)
  2. 用Evan的蓝色染色组织以评估膜稳定性的程度
    1. 在正常条件下种植植物。
    2. 施加应力处理以确定植物的特定生长阶段并收集组织(叶或根)。
      例如,我们强调从30天的花生植物收集的叶子。我们用1厘米直径的小叶盘进行叶盘测定。我们将10片叶片转移到培养皿中,然后施加甲基紫精(MV)诱导的氧化应激治疗。 5μM的甲基紫精暴露于一组(应力),另一组保存在纯水(对照)中,这些叶盘暴露于高光条件下(1000μmol/平方米) -1 )室温(RT)3小时。然后将这些叶片用Evan的蓝色染色剂染色(图1A)
    3. 在另一组实验中,NaCl(150mg,在Hoagland溶液中[参见方法2])对一组水稻幼苗(胁迫)施加压力,另一组在水培实验(Lopez)中仅保留在Hoagland's溶液(对照)中和Satti,1996)。压力强制三天后,从控制和应激植物收集根部,进行Evan蓝染色和膜稳定性测定(图1B)。该方法具有高达95%精度的高度可重现性
    4. 将收集的叶和根组织转移到2ml Eppendorf管中,并向每个管中加入2ml Evan's蓝色溶液。
      注意:调整音量以确保纸巾浸入溶液中。


      图1.使用Evan的蓝色染色测定膜稳定性。 (A)甲基紫精(MV)5μM处理的叶片和(B)150mM NaCl处理的根中的Evan蓝染色的显微镜检测。比例尺表示25μm。将来自30日龄植物的花生叶片在高光强度为1,000μmol/平方米以上的温度下温育3小时,随后叶片被Evan的蓝色染料染色。对于NaCl胁迫,将30天龄的水稻幼苗暴露于150mM NaCl 3天,并用Evan蓝染料染色以评估膜完整性。

    5. 以50振荡/分钟振荡振荡器振荡管20分钟 注意:抖动只是为了确保所有的组织都与Evan的蓝色染料溶液接触。
    6. 用蒸馏水彻底清洗根和叶子三次,或直到未结合的染料从表面洗出来 注意:(可选)根或叶组织也可以用pH 5.6的0.1 M CaCl 2 洗涤(参见配方3)
    7. 在正常透射光学显微镜下观察染色的叶或根。检查叶子或根,并在明场显微镜下拍摄图像。此外,Evan的蓝色染色图像可以使用imageJ软件进行量化,以评估细胞的活力。
      注意:如果样本量很大,最好只进行分光光度定量,并且可以应用适当的统计数据来达到显着性水平。
  3. 量化Evan由于膜不稳定而吸收的蓝色污点
    1. 从根部和叶子中提取Evan的蓝色染料,使用1ml 1%SDS(见配方4)细胞裂解缓冲液,其分解膜结构。
    2. 如果样本数量大,则使用每秒15次的组织淋巴细胞研磨100mg组织10分钟。如果只需要几个样品,就可以在杵和砂浆中研磨。研磨后,将提取物转移到2ml Eppendorf管中。
    3. 在室温下将提取物以7,168 x g离心5分钟,将染料洗脱至溶液中并除去碎屑。
    4. 将上清液转移到新管中或直接将250μl上清转移到96孔微量滴定板上。
      注意:由于Evan的蓝色染料不会氧化,因此可以在室温下储存半小时以测量大样品。
    5. 通过使用1%SDS作为空白,使用比色皿或平板读数器在600nm下以分光光度法测量光密度。
    6. Evan蓝色的浓度可以通过参考标准曲线来估计(图2)
    7. 绘制Evan蓝色浓度的图表,y轴和植物数量或x轴上的应力处理(图3A和3B)。


      图2. Evan蓝染色的标准图。 标准图是使用伊万蓝色储备溶液1μgml-sup。稀释不同浓度的埃文蓝,600nm处记录吸光度

      图3.使用分光光度法测定膜稳定性。 直方图显示了(A)叶组织和(B)根组织吸收的Evan蓝色染料的程度,作为膜损伤的反映。通过在1%十二烷基硫酸钠(SDS)中研磨组织并测量600nm处的吸光度并使用标准曲线图计算细胞吸收的染料的程度。错误栏表示三个重复数据的最小值。 *表示P 下的最小显着差异(LSD) 0.05使用GenStat统计工具分析。

  4. 使用Evan的蓝色准备标准曲线:
    将1μgEvan's蓝色溶解在1ml蒸馏水中,得到1μg/ ml的原液。从储备溶液中制成5个浓度,使体积达到1毫升。在分光光度计中在600nm处测量吸光度(图2)。

数据分析

使用至少三种生物重复来定量Evan蓝染料的摄取,并使用GenStat程序(Marcos等人,2013)分析数据。根据基因型和治疗水平的数量,使用方差分析(ANOVA)测试显着性水平。

食谱

  1. Evan的蓝色染色溶液
    将0.25g Evan's蓝色染料溶解于100ml pH 0.1的0.1M CaCl 2溶液中,并充分混合,直至溶解。 注意:Evan的蓝色解决方案应该每次新鲜准备。
  2. Hoagland解决方案
    本实验中使用的Hoagland溶液的组成在表1中给出。

    表1. Hoagland解决方案的组成

    *注意:在盐胁迫治疗中应避免这种情况。

  3. 0.1M CaCl 2 pH5.6的比例 为了制备0.1M CaCl 2,将1.10g CaCl 2(分子量= 110.989)溶于蒸馏水(1L)中,并用0.2N HCl将pH调节至5.6。 CaCl 2 2溶液可以在RT
    中储存一个月
  4. 1%SDS
    为了制备1%SDS,加入1g十二烷基硫酸钠至80ml蒸馏水中,并将其体积至100ml。使用前应先将SDS准备好。可选地,它可以在RT
    中存储一个月

致谢

印度农业研究理事会 - 利基卓越地区计划(F.10-(6)/ 2005 EPD)和(F. No. 10(15)2012 EPD)。 MUK承认支持印度NASI的铂金禧年奖学金。

参考

  1. Baker,CJ和Mock,NM(1994)。 改进的方法用于监测细胞悬浮液中的细胞死亡和使用Evan's蓝色的叶片测定。植物细胞组织器官培养 39(1):7-12。
  2. Gaff,DF和Okong'O-Ogola,O.(1971)。&lt; a class =“ke-insertfile”href =“https://academic.oup.com/jxb/article-abstract/22/3/ 756/575449 /用于测试的非渗透颜料 - 重定向的FANG = PDF“target =”_ blank“>使用非渗透色素测试细胞的存活。 J Exp Bot 22:756-758。
  3. Lopez,MV和Satti,SME(1996)。 Calcium和氯化钠胁迫下番茄钾增加和产量。植物科学114:19-27。
  4. Marcos,M.,Jean-Marcel,R.和Fred,AV(2013)。 多环境数据的统计分析:建模基因型与环境的相互作用及其遗传基础。前生理学4:44.
  5. Oprisko,MJ,Green,RL,Beard,JB和Gates,CE(1990)。 在12个温暖季节多年生草中的根毛的重要染色。作物科学 30:947-950。
  6. Vemanna,RS,Babitha,KC,Solanki,JK,Amarnatha Reddy,V.,Sarangi,SK和Udayakumar,M.(2017)。 Aldo-keto还原酶-1(AKR1)通过解毒反应性细胞毒性化合物来保护细胞酶免受盐胁迫。植物生理生化< / em> 113:177-186。
  7. Smith,BA,Reider,ML和Fletcher,JS(1982)。 植物组织培养物衰老期间生物染色与传代培养生长之间的关系植物生理学70:1228-1230。
  8. Sullivan,CY和Ross,WM(1979)。选择谷物高粱中的耐旱和耐热性在:Mussell,H.和Staples,RC (编辑)。作物植物应激生理学。 John Wiley and Sons pp:263-281。
  • English
  • 中文翻译
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: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:NV, P., PA, V., Vemanna, R. S., MS, S. and Makarla, U. (2017). Quantification of Membrane Damage/Cell Death Using Evan’s Blue Staining Technique. Bio-protocol 7(16): e2519. DOI: 10.21769/BioProtoc.2519.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。