A Protocol to Measure the Cytoplasmic Calcium in Arabidopsis Guard Cells
一种测定拟南芥保卫细胞中胞质钙离子的方法
发布: 2015年05月05日第5卷第9期 DOI: 10.21769/BioProtoc.1462 浏览次数: 11385
评审: Maria SinetovaAlexander JonesAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2014
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Abstract
Cytoplasmic calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca2+]cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca2+]cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca2+]cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.
Materials and Reagents
- Plant materials: Arabidopsis thaliana ecotype Columbia-0 (wild type) was obtained from ABRC at Ohio State University (Columbus)
- Glycine,N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-, (acetyloxy) methyl ester (Fluo-3, AM) (Life Technologies, InvitrogenTM, catalog number: F-14218 )
- Potassium chloride (KCl) (Sangon Biotech, catalog number: PB0440 )
- Calcium chloride dihydrate (CaCl2·2H2O) (Sangon Biotech, catalog number: C0556 )
- 2-(N-morpholino) ethanesulfonic acid (MES) (free acid, monohydrate) (Sigma-Aldrich, catalog number: M3671 )
- Potassium hydroxide (KOH) (Sangon Biotech, catalog number: PT1159 )
- Egtazic acid, Glycol ether diamine tetraacetic acid (EGTA) (Sangon Biotech, catalog number: ED007 )
- Chloroform (Sinopharm Chemical Reagent, catalog number: 10006818 )
- Sodium hypochlorite solution (Sangon Biotech, catalog number: S1944 )
- Agar (Sangon Biotech, catalog number: AJ637 )
- Murashige & Skoog (MS) Basal Medium w/Vitamins (PhytoTechnology Laboratories®, catalog number: M519 )
- Abscisic acid (ABA) (Sigma-Aldrich, catalog number: A1049 )
- Phosphatidic acid (PA) (Avanti Polar Lipid, catalog number: 840858C )
- Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
- Ethanol anhydrous (Sangon Biotech, catalog number: ET0737 )
- Tris (hydroxymethyl) aminomethane (Tris) (Sigma-Aldrich, catalog number: 252859 )
- Epidermal buffer (see Recipes)
- Fluo-3 AM stock solution (see Recipes)
- Loading buffer (see Recipes)
- ABA stock solution (see Recipes)
- ABA working solution (see Recipes)
- PA working solution (see Recipes)
Equipment
- Graduated centrifuge tubes (1.5 ml)
- Petri dish (6 cm diameter)
- Cover glass (20 mm * 20 mm, 0.13-0.17 μm thickness)
- Slides (76 mm * 26 mm, 1-2 mm thickness)
- Forceps
- Pipettor and matched tips
- Growth chamber (Percival, model: I-41LL )
- Zeiss LSM780 confocal laser scanning microscope
- Microprocessor pH meter (HANNA® Instruments, model: HI-2211 )
- Ultrasonic cell crusher (Scientz, model: JY92Ⅱ )
Software
- Zeiss confocal software ZEN 2012 (blue edition)
- Microsoft Excel
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Li, L., Lin, F., Qu, Y. and Zhang, Q. (2015). A Protocol to Measure the Cytoplasmic Calcium in Arabidopsis Guard Cells. Bio-protocol 5(9): e1462. DOI: 10.21769/BioProtoc.1462.
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分类
植物科学 > 植物生理学 > 离子分析
生物化学 > 其它化合物 > 离子 > 钙
细胞生物学 > 细胞成像 > 共聚焦显微镜
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