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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >ISOLATION OF PROTOPLASTS FOR PATCH-CLAMP EXPERIMENTS - AN IMPROVED METHOD REQUIRING MINIMAL AMOUNTS OF ADULT LEAF OR ROOT TISSUE FROM MONOCOTYLEDONOUS OR DICOTYLEDONOUS PLANTS
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ISOLATION OF PROTOPLASTS FOR PATCH-CLAMP EXPERIMENTS - AN IMPROVED METHOD REQUIRING MINIMAL AMOUNTS OF ADULT LEAF OR ROOT TISSUE FROM MONOCOTYLEDONOUS OR DICOTYLEDONOUS PLANTS

机译：斑点钳实验原生质体的分离-一种改良的方法，要求单子叶或双子叶植物的成年叶片或根组织的最小量

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For the purpose of patch-clamp studies, a protoplast isolation procedure is presented in which an osmotic shock (changing the osmolarity of the medium) and centrifugation step are omitted to limit mechanical stress. Apart from the reduction of mechanical stress factors, protoplast washing is also limited. Protoplasts have been isolated from different monocotyledonous and dicotyledonous plant species and from different tissues (leaves and roots). The seal success rate in patch-clamp experiments was high (about 85% successful seals showing a seal resistance > 10 G Omega). To evaluate the electrogenic viability of the protoplasts, fusicoccin and light responses of the plasma membrane and channel activity were tested. The addition of fusicoccin to the bathing medium caused typical high activation of the proton pump. Switching the light on and off caused transient depolarizations and hyperpolarizations, respectively, matching data reported for mesophyll cells in micro-electrode studies. Whole-cell and single-channel recordings of protoplast plasma membranes isolated from intact tobacco and Arabidopsis leaves were comparable with data published for protoplasts from corresponding tissue cultures. It is concluded that our isolation procedure yields protoplasts with electrogenic responses and is therefore suitable for patch-clamp studies in physiological research. [References: 19]
机译：为了进行膜片钳研究，提出了原生质体分离程序，其中省略了渗透压（改变介质的渗透压）和离心步骤以限制机械应力。除了减少机械应力因素外，原生质体洗涤也受到限制。已经从不同的单子叶和双子叶植物物种和不同的组织（叶和根）中分离出原生质体。膜片钳实验中的密封成功率很高（约有85％的成功密封显示出密封阻力> 10 G Omega）。为了评估原生质体的电生存能力，测试了融合霉素和质膜的光响应以及通道活性。在浴液中添加岩藻球菌素引起质子泵的典型高活化。打开和关闭灯分别导致瞬时去极化和超极化，与微电极研究中报告的叶肉细胞数据相匹配。从完整的烟草和拟南芥叶中分离的原生质体质膜的全细胞和单通道记录与来自相应组织培养的原生质体的数据相当。结论是，我们的分离程序可产生具有电响应的原生质体，因此适用于生理研究中的膜片钳研究。 [参考：19]

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