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Measurement of Endogenous H2O2 and NO and Cell Viability by Confocal Laser Scanning Microscopy
内源H2O2和NO以及保卫细胞活力的定量分析-共聚焦激光扫描显微镜联合荧光染料法   

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Abstract

Recently, there is compelling evidence that hydrogen peroxide (H2O2) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses including stomatal movement. Thus, the choice of sensitive methods for detection of endogenous H2O2 and NO in guard cells are very important for understanding the role of H2O2 and NO in guard cell signaling. In addition, besides stomatal closure caused by interfering guard cell signaling, it can also be caused by widespread, nonspecific damage to guard cells. To determine whether stomatal movement is caused by damage to guard cells, sensitive methods for detection of guard cell viability are often required.
The oxidatively sensitive fluorophore 2′,7′-dichlorofluorecin (H2DCF) is commonly employed to measure changes in intracellular H2O2 level directly. The non-polar diacetate ester (H2DCFDA) of H2DCF enters the cell and is hydrolysed into the more polar, non-fluorescent compound H2DCF, which, therefore, is trapped. Subsequent oxidation of H2DCF by H2O2, catalysed by peroxidases, yields the highly fluorescent DCF. Similarly, the cell-permeable, NO-sensitive fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2DA) is widely used for the direct detection of NO presence in both animal and plant cells. The non-polar DAF-2DA enters the cell and is hydrolyzed by cytosolic esterase into the more polar, non-fluorescent compound DAF-2, which in the presence of NO is converted to the highly fluorescent triazole derivative DAF-2T. The fluorescent indicator dyes fluorescein diacetate (FAD) and propidium iodide (PI) are widely used for detection of cell viability. FAD passes through cell membranes and is hydrolyzed by intracellular esterase to produce a polar compound that passes slowly through a living cell membrane but fast through a damaged or dead cell membrane, and thus accumulates inside the viable cells and exhibits green fluorescence when excited by blue light. In contrast, PI passes through damaged or dead cell membranes and intercalates with DNA and RNA to form a bright red fluorescent complex seen in the nuclei of dying or dead cells but not living cells. Based on the above analysis, the fluorescent indicator dyes H2DCFDA, DAF-2DA, FAD and PI load readily into guard cells, and their optical properties make them amenable to analysis by confocal laser scanning microscopy.
This protocol describes how to combine confocal laser scanning microscopy with fluorescent indicator dyes H2DCFDA, DAF-2DA, FAD and PI respectively for measurement of H2O2 and NO and viability of guard cell in leaves of Arabidopsis (Arabidopsis thaliana).

Materials and Reagents

  1. Leaves of Arabidopsis (Arabidopsis thaliana)
  2. Ethanesulfonic acid (MES)
  3. 2′,7′-dichlorofluorescin diacetate (H2DCFDA) (Sigma-Aldrich, catalog number: D6883 )
  4. 4, 5-diaminofluorescein diacetate (DAF-2DA) (Sigma-Aldrich, catalog number: D2813 )
  5. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4170 )
  6. Fluorescein diacetate (FDA) (Sigma-Aldrich, catalog number: F7378 )
  7. DMSO (Sigma-Aldrich, catalog number: D8418 )
  8. 10 mM MES/KCl buffer pH 6.15 (see Recipes)
  9. Tris/KCl buffer pH 7.2 (see Recipes)
  10. 10 mM H2DCFDA (see Recipes)
  11. 10 mM DAF-2DA (stock solution) (see Recipes)
  12. 1 mg/ml PI (stock solution) (see Recipes)
  13. 5 mg/ml FDA (stock solution) (see Recipes)

Equipment

  1. TCS-SP2 Confocal Laser Scanning Microscopy (Leica Lasertechnik Gmbh3, Heidelberg)
  2. 25 °C incubator
  3. Glass slide and cover glass
  4. Eyelbrow brush (Yellow wolf hair, length of hair: 0.8 cm, width of hair: 0.8 cm)
  5. Tweezers
  6. 6-cm diameter Petri plate

Software

  1. Leica confocal software (Leica Lasertechnik Gmbh3, Heidelberg)
  2. Photoshop software

Procedure

  1. Sampling.
    Arabidopsis seedlings were gown in plant growth chambers under a 16-h light/8-h dark cycle, a photon flux density of 0.1 mmol/m2/s, and a day/night temperature cycle of 18 °C/22 °C for 4-6 weeks. The youngest, fully expanded and flat leaves were harvested for immediate use.
  2. Opening the stomata.
    For stomatal closing experiments, to ensure stomata at fully opened stage before starting of treatments, the freshly harvested flat leaves were first floated with their abaxial surfaces facing up on MES/KCl buffer (15 ml) in 6-cm diameter Petri plates for 2-3 h at 22 °C under light condition (0.1 mmol/m2/s) to open the stomata, and then for subsequent treatments.
  3. Treating samples.
    Once the stomata were fully open (checked by microscope), the leaves were then floated on MES/KCl buffer alone or containing various compounds or inhibitors for required time at 22 °C under the same white light condition mentioned above or the desired conditions. Control treatments involved addition of buffer or appropriate solvents used with inhibitors.
    Note: As the epidermal strips is easier peeled from the abaxial surface than the paraxial surface of Arabidopsis leaves, we only peeled epidermal strips from abaxial surface of leaves for subsequent measurement. Thus, for treatments of UV-B radiation as well as other lights, to ensure the abaxial surface of leaves receiving same dose of UV-B radiation as well as other lights, the leaves were floated with their abaxial surfaces facing up and perpendicular to the light on MES/KCl buffer in all treatments including opening stomata.
  4. Peeling epidermal strips.
    After the above treatments, the leaf was taken out from MES/KCl buffer and a piece of filter paper was used to absorb the MES/KCl buffer on the surface of leaf. The leaf were flatly placed on a glass slide with its abaxial surfaces facing up, a tweezers was used to clamp a part of abaxial epidermis and mesophyll cells near the tip of leaf and the epidermal strips were quickly peeled along with the direction of the main leaf veins. Then, the peeled epidermal strips were immediately immersed in the corresponding treated buffer and pushed on the bottom of Petri plates by a forceps, the remained mesophyll cells were gently removed from epidermal strips by an eyebrow brush (Figure 1), and the tip of epidermal strip clamped by tweezers with more mesophyll cells was cut away, then epidermal strips were quickly used for loading of the fluorescent indicator dyes.
  5. Loading fluorescent indicator dyes.
    The peeled epidermal strips were immediately placed into Petri plates containing Tris-KCl buffer in the presence of H2DCFDA at a final concentration of 50 μM for 10 min, DAF-2DA at a final concentration of 10 mM for 30 min, FAD at a final concentration of 10 μg/ml for 10 min, or PI at a final concentration of 5 μg/ml for 10 min respectively, in the dark at 25 °C to exclude the possibility of that the fluorescent probes were oxidized or hydrolyzed by UV-B or PAR radiation. Then, the epidermal strips were washed with fresh Tris–KCl buffer without the fluorescent indicator dyes at least three times in dark to remove the excess dyes.
  6. Examination of H2O2, NO and viability of guard cells by confocal laser scanning microscopy. 
    After loading of the fluorescent indicator dyes, the slides were made and an examination of the peels was immediately performed by TCS-SP2 confocal laser scanning microscopy with the following settings: excitation 488 nm and emission 530 nm for H2DCFDA, DAF-2DA and FAD or excitation 536 nm and emission 617 nm for PI; normal scanning speed, frame 512 x 512. For example, by using these fluorescent indicator dyes and the confocal laser scanning microscope, Figure 1 clearly showed that guard cells of wild-type Arabidopsis under light alone had low levels of H2O2 (Figure 1A) and NO (Figure 1D), and high viability of guard cells (Figure 1G). However, 3 h of 0.5 W/m2 UV-B radiation significantly induced production of H2O2 (Figure 1B) and NO (Figure 1E), and did not affect cell viability (Figure 1H) in wild-type guard cells, but did not induce H2O2 production in guard cells of AtrbohD/F double mutant (Figure 1C) or NO production in guard cells of Nia1-2/Nia2-5 double mutant (Figure 1F). Furthermore, when wild-type leaves were exposed to 0.8 W/m2 UV-B for 3 h, the guard cells were significantly damaged and clearly marked by the fluorescent dye PI (Figure 1I).
  7. Analysis
    Images acquired from the confocal microscope were analyzed with Leica confocal software to measure the average fluorescent pixel intensities in the guard cells following various treatments (such as in Figure 1J; the detailed procedure of analysis seen the following note) and processed with Photoshop software. In each experiment, three epidermal strips were at least measured, each of which originated from a different plant. Each experiment was repeated three times. The selected confocal image represented the same results from approximately nine time measurements. Data of fluorescence pixel intensities are statistically analyzed by one-way ANOVA and displayed as means ± SE (n = 60).
    Note: Procedure for analysis of fluorescent intensity: On the “LAS AF” screen of Leica confocal software, click the “quality” button, select “Histogram” analysis method and circle guard cell to be analyzed, then “Statistics” shows the average fluorescent intensity of the circled guard cell, select “Export as” to save the “Statistics” displayed data in a text format.


    Figure 1. Effects of UV-B radiation on the production of H2O2 and NO and viability of Arabidopsis guard cells. A-C. Images of guard cells loaded with the fluorescent indicator dye H2DCFDA. D-F. Images of guard cells loaded with the fluorescent indicator dye DAF-2DA. G and H. Images of guard cells loaded with the fluorescent indicator dye FAD. A, D and G. Wild-type guard cells exposed to light alone for 3 h. B, E and H. Wild-type guard cells exposed to light with 0.5 W m-2 for 3 h. C and F. Double mutants AtrbohD/F and Nia1-2/Nia2-5 guard cells respectively exposed to light with 0.5 W m-2 for 3 h. I. Image of wild-type guard cells exposed to 0.8 W m-2 UV-B for 3 h and loaded with the fluorescent indicator dye PI. J. The figure shows the average fluorescent intensities (means ± SE) of guard cells in images from A to H. The guard cells shown in images a-i are representative of guard cells shown in images A-I, respectively. Scale bars in image I (75 μm) and i (25 μm) are for images A-I and a-i, respectively.

Recipes

  1. 10 mM MES/KCl buffer (10 mM MES, 50 mM KCl, 0.1 mM CaCl2, pH 6.15, 500 ml)
    1.066 g MES
    5.549 mg CaCl2
    1.86375 g KCl
    Mix these chemicals with 400 ml dH2O
    Adjust pH to 6.15 with KOH
    Add dH2O to 500 ml
    Stored at room temperature
  2. Tris/KCl buffer (10 mM Tris and 50 mM KCl, pH 7.2, 500 ml)
    0.6055 g Tris
    1.86 g KCl
    Mix these chemicals with 400 ml dH2O
    Adjust pH to 7.2 with HCl
    Add dH2O to 500 ml
    Stored at room temperature
  3. 10 mM H2DCFDA (1 ml, stock solution).
    Mix 4.8729 mg of H2DCFDA with 1 ml DMSO
    Stored at -20 °C
    This stock solution is diluted by Tris/KCl pH 7.2 buffer to get a working concentration of 50 μM
  4. 10 mM DAF-2DA (stock solution).
    Mix 1 mg of DAF-2DA with 224 μl DMSO to form 10 mM stock solution
    Stored at -20 °C
    This stock solution is diluted by Tris/KCl pH 7.2 buffer to get a working concentration of 10 μM
  5. 1 mg/ml PI (stock solution)
    Mix 1 mg of PI with 1 ml dH2O to make a stock solution
    Stored at 4 °C in a dark bottle
    This stock solution is diluted by Tris/KCl pH 7.2 buffer to get a working concentration of 5 μg/ml
  6. 5 mg/ml FDA (stock solution)
    Mix 5 mg of FDA with 1 ml acetone to make a stock solution
    Stored at 4 °C in a dark bottle
    This stock solution is diluted by Tris/KCl pH 7.2 buffer to get a working concentration of 10 μg/ml

Acknowledgments

This work was supported by the National Science Foundation of China (grant no. 31170370) and the Fundamental Research Funds for the Central Universities (grant no. GK200901013). This protocol was adapted from previously published paper He et al. (2013).

References

  1. He, J. M., Ma, X. G., Zhang, Y., Sun, T. F., Xu, F. F., Chen, Y. P., Liu, X. and Yue, M. (2013). Role and interrelationship of Galpha protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiol 161(3): 1570-1583.

简介

最近,有令人信服的证据表明,过氧化氢(H 2 O 2 O 2)和一氧化氮(NO)作为植物中的信号分子起作用,介导一系列反应,包括气孔运动。因此,选择用于检测保卫细胞中内源性H 2 O 2 O 2和NO的灵敏方法对于理解H 2 sub的作用是非常重要的> O 2和保守细胞信号中的NO。此外,除了由干扰保卫细胞信号传导引起的气孔闭合之外,它还可以由保护细胞的广泛的非特异性损伤引起。为了确定气孔运动是否由保卫细胞的损伤引起,通常需要用于检测保卫细胞存活力的灵敏方法。
氧化敏感荧光团2',7'-二氯荧光素(H 2 DCF)通常用于测量细胞内H 2 O 2 O 2的变化,/sub>级别。 H 2 DCF的非极性二乙酸酯(H 2 DCFDA)进入细胞,并水解成极性更强的非荧光化合物H 2,/sub> DCF,因此,它被捕获。随后通过过氧化物酶催化的H 2 O 2 O 2对H 2 DCF的氧化产生高度荧光的DCF。类似地,细胞可渗透的NO-敏感性荧光探针4,5-二氨基荧光素二乙酸酯(DAF-2DA)广泛用于在动物和植物细胞中直接检测NO存在。非极性DAF-2DA进入细胞,并被胞质酯酶水解为极性更强的非荧光化合物DAF-2,其在NO存在下转化为高度荧光的三唑衍生物DAF-2T。荧光指示剂染料荧光素二乙酸酯(FAD)和碘化丙啶(PI)广泛用于检测细胞活力。 FAD通过细胞膜并被胞内酯酶水解以产生极性化合物,其缓慢通过活细胞膜但快速通过受损或死亡的细胞膜,并因此积聚在活细胞内并且当被蓝光激发时显示绿色荧光。相反,PI通过损伤的或死亡的细胞膜并且与DNA和RNA插入以形成在死亡或死亡细胞而非活细胞的细胞核中看到的亮红色荧光复合物。基于上述分析,荧光指示剂染料H 2 2 DCFDA,DAF-2DA,FAD和PI容易负载到保卫细胞中,并且它们的光学性质使其适于通过共聚焦激光扫描显微镜进行分析。该协议描述了如何将共焦激光扫描显微镜与荧光指示剂染料H sub 2 DCFDA,DAF-2DA,FAD和PI分别组合用于测量H 2 O 2 - 拟南芥(拟南芥)的叶中的保护细胞的生存力和潜力。

材料和试剂

  1. 拟南芥(拟南芥)的叶子
  2. 乙磺酸(MES)
  3. 2',7'-二氯荧光素二乙酸酯(H 2 DCFDA)(Sigma-Aldrich,目录号:D6883)
  4. (DAF-2DA)(Sigma-Aldrich,目录号:D2813)
  5. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4170)
  6. 荧光素二乙酸酯(FDA)(Sigma-Aldrich,目录号:F7378)
  7. DMSO(Sigma-Aldrich,目录号:D8418)
  8. 10mM MES/KCl缓冲液pH6.15(见配方)
  9. Tris/KCl缓冲液pH 7.2(参见配方)
  10. 10mM H 2 DCFDA(参见配方)
  11. 10mM DAF-2DA(储备溶液)(见配方)
  12. 1 mg/ml PI(储备溶液)(见配方)
  13. 5 mg/ml FDA(储存液)(参见配方)

设备

  1. TCS-SP2共聚焦激光扫描显微镜(Leica Lasertechnik Gmbh3,Heidelberg)
  2. 25℃培养箱
  3. 玻璃滑盖和盖玻璃
  4. 眼眉刷(黄狼毛,头发长度:0.8cm,头发宽度:0.8cm)
  5. 镊子
  6. 直径6cm的培养皿

软件

  1. Leica共焦软件(Leica Lasertechnik Gmbh3,Heidelberg)
  2. Photoshop软件

程序

  1. 取样。
    将拟南芥幼苗在16小时光照/8小时黑暗循环下在植物生长室中长成,光子通量密度为0.1mmol / m 2 /s,昼/夜温度周期为18°C/22°C 4-6周。收获最年轻,完全展开和扁平的叶子以供立即使用
  2. 打开气孔。
    对于气孔关闭实验,为了在开始处理之前确保气孔处于完全打开的阶段,首先将新鲜收获的平叶漂浮,使其背面朝上在6cm直径培养皿中的MES/KCl缓冲液(15ml)在光照条件下在22℃下3小时(0.1mmol / m 2/s/s)以打开气孔,然后用于随后的处理。
  3. 处理样品。
    一旦气孔完全打开(通过显微镜检查),然后将叶子在单独的MES/KCl缓冲液上漂浮或在22℃下在上述相同的白光条件或所需的条​​件下漂浮在含有各种化合物或抑制剂所需的时间。控制处理涉及添加缓冲液或与抑制剂一起使用的适当溶剂 注意:由于表皮条比拟南芥叶的近轴表面更容易从后轴表面剥离,我们只从叶的后表面剥离表皮条用于随后的测量。因此,对于UV-B辐射以及其它光的处理,为了确保接受相同剂量的UV-B辐射的叶子的背面表面以及其它光,叶子浮动,它们的背面朝上并垂直于所有处理中的光照在MES/KCl缓冲液上,包括打开气孔。
  4. 剥皮表皮条。
    在上述处理之后,从MES/KCl缓冲液中取出叶子,并使用一片滤纸吸收叶子表面上的MES/KCl缓冲液。将叶片平放在载玻片上,其背面朝上,用镊子夹住叶尖附近的背轴表皮和叶肉细胞的一部分,并且将表皮条带随主叶的方向快速剥离静脉。然后,将剥离的表皮条立即浸入相应的处理缓冲液中,并通过镊子推到培养皿的底部,通过眉毛刷轻轻地从表皮条上除去剩余的叶肉细胞(图1),并且将表皮尖切除带有更多叶肉细胞的镊子夹紧的条带,然后快速使用表皮条带装载荧光指示剂染料。
  5. 加载荧光指示剂染料 将剥离的表皮条立即置于含有Tris-KCl缓冲液的培养皿中,在H 2 DCFDA存在下,终浓度为50μM,10分钟,DAF-2DA,终浓度为10mM 30分钟,在10μg/ml的终浓度的FAD 10分钟或在5μg/ml的终浓度的PI分别10分钟,在25℃的黑暗中,以排除荧光的可能性探针被UV-B或PAR辐射氧化或水解。然后,在没有荧光指示剂染料的新鲜Tris-KCl缓冲液中在黑暗中将表皮条带洗涤至少三次以除去过量的染料。
  6. 通过共聚焦激光扫描显微镜检查H 2 O 2亚基,保护细胞的存活率和保持细胞的存活率。
    在加载荧光指示剂染料后,制备载玻片,并立即通过TCS-SP2共聚焦激光扫描显微镜进行剥离检查,其具有以下设置:H sub 2的激发488nm和发射530nm > DCFDA,DAF-2DA和FAD或PI的激发536nm和发射617nm;正常扫描速度,框架512×512.例如,通过使用这些荧光指示剂染料和共聚焦激光扫描显微镜,图1清楚地显示,在光照下单独的野生型拟南芥的保卫细胞具有低水平(图1A)和NO(图1D),以及保护细胞的高存活率(图1G)。然而,3小时的0.5W/m 2的UV-B辐射显着诱导H 2 O 2(图1B)和NO(图1B)的产生,图1E),并且不影响野生型保卫细胞中的细胞活力(图1H),但在的保卫细胞中不诱导H 2 O 2 O 2 > Nia1-2/Nia2-5双突变体(图1F)的保卫细胞中的AtrbohD/F(AtrbohD/F)双突变体(图1C)或NO产生。此外,当野生型叶暴露于0.8W/m 2 UV-B 3小时时,保卫细胞显着受损并被荧光染料PI清楚地标记(图1I)。
  7. 分析
    使用Leica共聚焦软件分析从共聚焦显微镜获取的图像,以测量在各种处理(例如图1J;详述的分析程序见下文)中并用Photoshop软件处理后的保卫细胞中的平均荧光像素强度。在每个实验中,至少测量三个表皮条,每个来自不同的植物。每个实验重复三次。所选择的共焦图像表示来自大约九次时间测量的相同结果。通过单因素方差分析统计分析荧光像素强度的数据并显示为平均值±SE(n = 60)。
    注意:分析荧光强度的步骤:在Leica共焦软件的"LAS AF"屏幕上,单击"质量"按钮,选择"直方图"分析方法和要分析的圆形保护单元格,然后"统计"显示圆形保护单元的平均荧光强度,选择"导出为"以文本格式保存"统计"显示的数据。


    图1. UV-B辐射对H 2 O 2 A-C。 担载有荧光指示剂染料H 2 DCFDA的保卫细胞的图像。 D-F。保卫细胞的图像装载有荧光指示剂染料DAF-2DA。 G和H.带有荧光指示剂染料FAD的保卫细胞的图像。 A,D和G.野生型保卫细胞单独暴露3小时。 B,E和H。野生型保卫细胞用0.5W m -2 - 光照射3小时。 C和F双重突变体分别使用0.5W m -2 -/- 上清液分别暴露于光下的AtrbohD/F和em1N1-2/Nia2-5/3小时。 I.暴露于0.8W m -2 -2UV-B 3小时并装载有荧光指示剂染料P1的野生型保卫细胞的图像。该图显示了从A至H的图像中保护细胞的平均荧光强度(平均值±SE)。图像a-i中所示的保护细胞分别代表图像A-1中所示的保护细胞。图像I(75μm)和i(25μm)中的比例尺分别用于图像A-I和a-i。

食谱

  1. 10mM MES/KCl缓冲液(10mM MES,50mM KCl,0.1mM CaCl 2,pH 6.15,500ml)中。
    1.066克MES
    5.549mg CaCl 2
    1.86375克KCl
    将这些化学品与400ml dH 2 O混合 用KOH将pH调节至6.15 将dH <2> O添加到500ml
    在室温下贮存
  2. Tris/KCl缓冲液(10mM Tris和50mM KCl,pH7.2,500ml) 0.6055克Tris
    1.86克KCl
    将这些化学品与400ml dH 2 O混合 用HCl
    调节pH至7.2 将dH <2> O添加到500ml
    在室温下贮存
  3. 10mM H 2 DCFDA(1ml,储备溶液) 将4.8729mg H 2 DCFDA与1ml DMSO混合 储存于-20°C
    该储备溶液用Tris/KCl pH7.2缓冲液稀释,得到50μM的工作浓度
  4. 10mM DAF-2DA(储备溶液)。
    将1mg DAF-2DA与224μlDMSO混合以形成10mM储备溶液
    储存于-20°C
    该储备溶液用Tris/KCl pH7.2缓冲液稀释,得到10μM的工作浓度
  5. 1 mg/ml PI(储备液)
    将1mg PI与1ml dH 2 O混合以制备储备溶液
    储存在4℃的黑暗瓶子中
    该储备溶液用Tris/KCl pH7.2缓冲液稀释,得到5μg/ml的工作浓度
  6. 5 mg/ml FDA(储备液)
    将5mg FDA与1ml丙酮混合,制成储备溶液 储存在4℃的黑暗瓶子中
    该储备溶液用Tris/KCl pH7.2缓冲液稀释,得到10μg/ml的工作浓度

致谢

这项工作得到中国国家科学基金会(拨款号31170370)和中央大学基础研究基金(拨款号GK200901013)的支持。 该协议改编自以前发表的论文He (2013)。

参考文献

  1. He,J. M.,Ma,X. G.,Zhang,Y.,Sun,T. F.,Xu,F. F.,Chen,Y. P.,Liu,X.和Yue, Galpha蛋白,过氧化氢和一氧化氮在紫外线B诱导的气孔关闭中的作用和相互关系 植物生理 161(3):1570-1583。
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  • 中文翻译
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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. Wu, M., Ma, X. and He, J. (2013). Measurement of Endogenous H2O2 and NO and Cell Viability by Confocal Laser Scanning Microscopy. Bio-protocol 3(19): e920. DOI: 10.21769/BioProtoc.920.
  2. He, J. M., Ma, X. G., Zhang, Y., Sun, T. F., Xu, F. F., Chen, Y. P., Liu, X. and Yue, M. (2013). Role and interrelationship of Galpha protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiol 161(3): 1570-1583.
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Ren Bing
Northwest A & F University
In you protocol, you make the H2DCFDA and DAF-2DA stock solution by the organic solvent DMSO, but make working solution by the diluent Tris-HCl.Could you tell me the reason that you use the two different solvent and the effects on the detection results? Thank you very much!
12/10/2013 5:06:18 PM Reply
Junmin He
School of Life Sciences, Shaanxi Normal University, China

The two probes are soluble in organic solvents such as DMSO and will be stable for at least six months in these solvents if stored at -20C, but are sparingly soluble in aqueous buffers. For use, the stock solution can be immediately diluted with buffer to achieve the desired concentration or pH, but these working solutions are not stable and not recommend storing for more than one day. So, we use the two different solvent for stock solution and the working sdolution. To exclude the effect of DMSO on the stomatal aperture or the fluorescence intensities, we make control treatments involved the addition of DMSO at the same concentration in the working solution but not involved the fluorescent probe, the results showed that the DMSO at the concentration in working solution had no effect either on the stomatal aperture or the fluorescence.

12/17/2013 4:59:07 PM