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Increasing the Membrane Permeability of a Fern with DMSO
使用DMSO增加膜蕨类的膜通透性   

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Plant Biology
Nov 2017

 

Abstract

Cell membrane prevents the entrance of extra molecules (e.g., transcription and translation inhibitors) into the cell. For studying the physiological effects of transcription and translation inhibitors on Hymenophyllum caudiculatum fronds, we incubate fronds with 0.1% DMSO to test if this increases cell membrane permeability relative to incubation with ultrapure water. The study showed that DMSO could significantly improve the cell membrane permeability of filmy fronds.

Keywords: Filmy ferns (膜蕨类), Membrane permeability (膜通透性), DMSO (DMSO), Propidium iodide (碘化丙啶)

Background

One of the most remarkable characteristics of filmy ferns is that the frond, apart from the vascular tissue, is made of a single cell layer and lacks stomata. In our previous work (Garcés et al. 2018), we incubated Hymenophyllum caudiculatum fronds with cycloheximide or actinomycin D to study the effects of translation, or transcription inhibition respectively. If an exogen inhibitor incubated with a filmy fern frond does not affect plant physiology, it may be because the inhibitor fails to enter the cell. In order to improve the entrance of inhibitors into the cell, we tested if an aqueous solution of 0.1% DMSO has effects on cell membrane permeability, visualizing the entrance of propidium iodide (PI) into the cells.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes
  2. Pipette tips
  3. Plain slides 75 x 25 mm (VWR, catalog number: 48300-026 )
  4. Micro cover slides, square 22 x 22 mm (VWR, catalog number: 48366-067 )
  5. Filter (0.22 μm)
  6. DMSO (Sigma-Aldrich, catalog number: D8418 )
  7. Propidium iodide (PI) 10 mg (Sigma-Aldrich, catalog number: P4170 )
  8. Ultrapure water 18.2 MΩcm
  9. PI (Propidium Iodide) stock (see Recipes)
  10. PI-H2O control (see Recipes)
  11. PI-DMSO solution (see Recipes)

Equipment

  1. Pipettes
  2. Confocal laser scanning microscope (Olympus, model: Fluoview FV1000 )
  3. Ultrapure water system (Elga LabWater, model: PureLab Classic )

Software

  1. FV10ASW v.2.0c
  2. ImageJ 2.0.0-rc-41/1.50d

Procedure

  1. Performing the permeability test in 1.5 ml microcentrifuge tubes
    1. Add 1 ml PI-DMSO solution (Recipe 3) and PI-H2O control (Recipe 2) into different 1.5 ml microcentrifuge tubes. Put freshly obtained, similar size about 1 cm of pinnae into the tubes. Incubate at room temperature.
    2. After 5 min, 30 min, 2 h, and 5 h take out the pinnae, rinse briefly in distilled water to remove residual PI, and then mount on slides to observe fluorescence under Confocal microscope.
  2. Confocal microscopy
    1. Observe the Intracellular PI fluorescence under a Fluoview FV1000 confocal laser scanning Biological Microscope (Olympus, Japan). The software FV10ASW v.2.0c and ImageJ 2.0.0-rc-41/1.50d are used to capture the images and construct Z projections. Detect chlorophyll autofluorescence using excitation and emission wavelengths of 488 nm and 687 nm, respectively, PI using excitation and emission wavelengths of 538 and 619 nm, respectively.
    2. We found that after 30 min of incubation, the PI had entered the cells (Figure 1). Images were obtained from three samples.


      Figure 1. Permeability test for Hymenophyllum caudiculatum fronds. Hymenophyllum caudiculatum fronds were incubated with 0.1% DMSO or distilled water (control) with 1.5 mM PI. Fluorescence of the chlorophyll (left panel) or PI (right panel) is observed. The same field of view was recorded at different wavelengths.

Data analysis

Three samples were analyzed and representative results are shown in Figure 1. After 30 min of incubation with 0.1% DMSO, PI fluorescence starts being visible inside the cells and reaches the maximum after 5 h. In contrast, PI fluorescence was almost invisible after 5 h incubation of PI with ultrapure water.

Recipes

  1. PI (Propidium Iodide) stock solution (1 mg/ml or 1.5 mM)
    To prepare, add 10ml distilled water to 10 mg PI. Store 1 ml aliquots in 1.5 ml tubes at -20 °C, protecting from light
  2. PI-H2O control (10 ml)
    1.5 μM propidium Iodide
    ultrapure water 
    1. In 10 ml of ultrapure water add 10 μl of 1.5 mM PI (stock solution)
    2. Filter sterilize (0.22 μm) and store at room temperature
  3. PI-DMSO solution (10 ml)
    0.1% DMSO
    1.5 μM propidium Iodide
    1. In 10 ml of ultrapure water add 10 μl of DMSO and 10 μl of 1.5 mM PI (stock solution)
    2. Filter sterilize (0.22 μm) and store at room temperature

Acknowledgments

This work was supported by grant #11130717 from FONDECYT. The author declares no conflicts of interest or competing interests.

References

  1. Garcés, M., Ulloa, M., Miranda, A. and Bravo, L. A. (2018). Physiological and ultrastructural characterisation of a desiccation-tolerant filmy fern, Hymenophyllum caudiculatum: Influence of translational regulation and ABA on recovery. Plant Biol Mar 20(2): 288-295.

简介

细胞膜防止额外分子(例如,转录和翻译抑制剂)进入细胞。 为了研究转录和翻译抑制剂对Hymenophyllum caudiculatum叶的生理影响,我们将叶用0.1%DMSO温育以测试它是否相对于用超纯水孵育增加细胞膜渗透性。 该研究表明DMSO可显着提高膜状叶的细胞膜透性。

【背景】薄膜蕨类植物最显着的特征之一是,除了维管组织外,叶子由单细胞层组成,缺少气孔。 在我们以前的工作中(Garcéset al。 2018年),我们用放线菌酮或放线菌素D孵育了Hymenophyllum caudiculatum叶,分别研究翻译或转录抑制的作用。 如果与薄膜蕨类植物一起孵育的外源抑制剂不影响植物生理,可能是因为抑制剂不能进入细胞。 为了改善抑制剂进入细胞,我们测试了0.1%DMSO水溶液是否对细胞膜通透性有影响,观察到碘化丙啶(PI)进入细胞。

关键字:膜蕨类, 膜通透性, DMSO, 碘化丙啶

材料和试剂

  1. 1.5 ml微量离心管
  2. 移液器吸头
  3. 平板幻灯片75 x 25毫米(VWR,目录号:48300-026)
  4. 微盖玻片,方形22 x 22毫米(VWR,目录号:48366-067)
  5. 过滤器(0.22微米)
  6. DMSO(Sigma-Aldrich,目录号:D8418)
  7. 碘化丙锭(PI)10mg(Sigma-Aldrich,目录号:P4170)
  8. 超纯水18.2MΩcm
  9. PI(Propidium Iodide)库存(见食谱)
  10. PI-H 2 O对照(见食谱)
  11. PI-DMSO溶液(见食谱)

设备

  1. 移液器
  2. 共聚焦激光扫描显微镜(奥林巴斯,型号:Fluoview FV100)
  3. 超纯水系统(Elga LabWater,型号:PureLab Classic)

软件

  1. FV10ASW v.2.0c
  2. ImageJ 2.0.0-rc-41 / 1.50d

程序

  1. 在1.5ml微量离心管中进行渗透性测试
    1. 加入1ml PI-DMSO溶液(配方3)和PI-H 2 O对照(配方2)到不同的1.5ml微量离心管中。新鲜获得,约1厘米的羽片相似的大小进入管。在室温下孵育。
    2. 5分钟,30分钟,2小时和5小时后取出耳廓,在蒸馏水中短暂冲洗以去除残留的PI,然后在共聚焦显微镜下安装在载玻片上以观察荧光。
  2. 共焦显微镜
    1. 在Fluoview FV1000共聚焦激光扫描生物显微镜(奥林巴斯,日本)下观察细胞内PI荧光。软件FV10ASW v.2.0c和ImageJ 2.0.0-rc-41 / 1.50d用于捕获图像并构建Z投影。使用激发和发射波长分别为488 nm和687 nm的PI检测叶绿素自发荧光,PI分别使用538和619 nm的激发和发射波长。
    2. 我们发现孵育30分钟后,PI已进入细胞(图1)。图像来自三个样本。


      图1. Hymenophyllum caurdulatum叶的渗透性测试。将Hymenophyllum caudiculatum叶与0.1%DMSO或蒸馏水(对照)一起用1.5mM PI 。观察叶绿素(左图)或PI(右图)的荧光。
      在不同的波长记录相同的视野。

数据分析

分析三个样品并且代表性结果显示在图1中。在用0.1%DMSO温育30分钟后,PI荧光在细胞内开始可见并且在5小时后达到最大值。相反,PI与超纯水孵育5小时后PI荧光几乎不可见。

食谱

  1. PI(Propidium Iodide)储备液(1 mg / ml或1.5 mM)
    准备时,加10毫升蒸馏水至10毫克PI。将1毫升等分试样储存在-20℃的1.5毫升试管中,避光保存。
  2. PI-H 2 O对照(10ml)

    1.5μM碘化丙锭 超纯水 
    1. 在10 ml超纯水中加入10μl1.5 mM PI(原液)
    2. 过滤消毒(0.22μm)并在室温下储存
  3. PI-DMSO溶液(10毫升)
    0.1%DMSO
    1.5μM碘化丙锭
    1. 在10ml超纯水中加入10μlDMSO和10μl1.5mM PI(储备液)
    2. 过滤消毒(0.22μm)并在室温下储存

致谢

这项工作得到了来自FONDECYT的资助#11130717的支持。作者声明不存在利益冲突或利益冲突。

参考

  1. Garcés,M.,Ulloa,M.,Miranda,A.和Bravo,L.A。(2018)。 耐干旱的薄膜蕨类植物的生理和超微结构表征,Hymenophyllum caudiculatum :翻译调控和ABA对恢复的影响。 植物生物学期刊 20(2):288-295。
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引用:Garcés, M. (2018). Increasing the Membrane Permeability of a Fern with DMSO. Bio-protocol 8(12): e2896. DOI: 10.21769/BioProtoc.2896.
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