Name: staining the cytoplasmic ca2+ with fluo-4/am in apple pulp
Uploaded: 2021-11-06T13:00:00
Description: Watch this Scientific Journal Video about Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp at JoVE.com

这项工作得到了山东省农业品种改良项目（2019LZGC007）和山东现代农业产业技术体系果树创新团队（SDAIT-06-05）的支持。

1. 前列座提取
<ol><li>准备基本解决方案：20 mM CaCl 2，5mM 2-（N-形态）乙醇酸和0.4M D-索比托。 注：基本溶液的 pH 值调整为 5.8，带有 0.1 M Tris 缓冲器，通过 0.22 μm 水溶性滤清器过滤，并存储在 4 °C 下。</li>
	<li>准备酶溶液：将 0.3%（w/v） 马塞罗酶 R-10 和 0.5%（w/v） 纤维酶 R-10 与基本溶液混合。</li>
	<li>将 0.5 mL 的酶溶液加入 1.5 mL 离心机管中。挑选一个健康成熟的苹果。然后...

<ol>
	<li>Hocking, B., Tyerman, S. D., Burton, R. A., Gilliham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+calcium:+Transport+and+physiology.">Fruit calcium: Transport and physiology.</a> Frontiers in Plant Science. 7, 569 (2016).</li><li>Li, J., Yang, H. -. q., Yan, T. -. l., Shu, H. -. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+indole+butyric+acid+on+the+transportation+of+stored+calcium+in+Malus+hupehensis+rhed.+Seedling.">Effect of indole butyric acid on the transportation of stored calcium in Malus hupehensis rhed. Seedling.</a> Agricultural Sciences in China. 5 (11), 834-838 (2006).</li><li>Gao, Q., Xiong, T., Li, X., Chen, W., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+calcium+sensors+in+fruit+development+and+ripening.">Calcium and calcium sensors in fruit development and ripening.</a> Scientia Horticulturae. 253, 412-421 (2019).</li><li>Barrett, D. M., Beaulieu, J. C., Shewfelt, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color,+flavor,+texture,+and+nutritional+quality+of+fresh-cut+fruits+and+vegetables:+desirable+levels,+instrumental+and+sensory+measurement,+and+the+effects+of+processing.">Color, flavor, texture, and nutritional quality of fresh-cut fruits and vegetables: desirable levels, instrumental and sensory measurement, and the effects of processing.</a> Critical Reviews in Food Science and Nutrition. 50 (5), 369-389 (2010).</li><li>Deell, J. R., Khanizadeh, S., Saad, F., Ferree, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+apple+fruit+firmness--a+review.">Factors affecting apple fruit firmness--a review.</a> Journal- American Pomological Society. 55 (1), 8-27 (2001).</li><li>Johnston, J., Hewett, E., Hertog, M. A. T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postharvest+softening+of+apple+(Malus+domestica)+fruit:+A+review.">Postharvest softening of apple (Malus domestica) fruit: A review.</a> New Zealand Journal of Experimental Agriculture. 30 (3), 145-160 (2002).</li><li>Demidchik, V., Shabala, S., Isayenkov, S., Cuin, T. A., Pottosin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transport+across+plant+membranes:+mechanisms+and+functions.">Calcium transport across plant membranes: mechanisms and functions.</a> New Phytologist. 220 (1), 49-69 (2018).</li><li>Miqueloto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+regulating+fruit+calcium+content+and+susceptibility+to+bitter+pit+in+cultivars+of+apple.">Mechanisms regulating fruit calcium content and susceptibility to bitter pit in cultivars of apple.</a> Acta horticulturae. 1194 (1194), 469-474 (2018).</li><li>Kou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+CaCl2+dipping+and+pullulan+coating+on+the+development+of+brown+spot+on+'Huangguan'+pears+during+cold+storage.">Effects of CaCl2 dipping and pullulan coating on the development of brown spot on 'Huangguan' pears during cold storage.</a> Postharvest Biology and Technology. 99, 63-72 (2015).</li><li>Vinh, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+on+blossom-end+rot+incidence+in+two+tomato+cultivars+in+relation+to+calcium+nutrition+and+fruit+growth.">Comparative analysis on blossom-end rot incidence in two tomato cultivars in relation to calcium nutrition and fruit growth.</a> The Horticulture Journal. 87 (1), 97-105 (2018).</li><li>Yamane, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foliar+calcium+applications+for+controlling+fruit+disorders+and+storage+life+in+deciduous+fruit+trees.">Foliar calcium applications for controlling fruit disorders and storage life in deciduous fruit trees.</a> Japan Agricultural Research Quarterly. 48 (1), 29-33 (2014).</li><li>Grienberger, C., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+in+neurons.">Imaging calcium in neurons.</a> Neuron. 73 (5), 862-885 (2012).</li><li>Bootman, M. D., Rietdorf, K., Collins, T. J., Walker, S., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-sensitive+fluorescent+dyes+and+intracellular+Ca2++imaging.">Ca2+-sensitive fluorescent dyes and intracellular Ca2+ imaging.</a> CSH Protocols. 2013 (2), 83 (2013).</li><li>Hirabayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+practical+red+fluorescent+probe+for+cytoplasmic+calcium+ions+with+greatly+improved+cell-membrane+permeability.">Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.</a> Cell Calcium. 60 (4), 256-265 (2016).</li><li>Krebs, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET-based+genetically+encoded+sensors+allow+high-resolution+live+cell+imaging+of+Ca(2)(+)+dynamics.">FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca(2)(+) dynamics.</a> Plant Journal. 69 (1), 181-192 (2012).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+palette+of+genetically+encoded+Ca(2)(+)+indicators.">An expanded palette of genetically encoded Ca(2)(+) indicators.</a> Science. 333 (2), 1888-1891 (2011).</li><li>Gee, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physiological+characterization+of+fluo-4+Ca2+-indicator+dyes.">Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes.</a> Cell Calcium. 27 (2), 97-106 (2000).</li><li>Qu, H., Xing, W., Wu, F., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+inexpensive+method+of+loading+fluorescent+dye+into+pollen+tubes+and+root+hairs.">Rapid and inexpensive method of loading fluorescent dye into pollen tubes and root hairs.</a> PLoS One. 11, 0152320 (2016).</li><li>Suwi&#324;ska, A., Was&#261;g, P., Zakrzewski, P., Lenartowska, M., Lenartowski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calreticulin+is+required+for+calcium+homeostasis+and+proper+pollen+tube+tip+growth+in+Petunia.">Calreticulin is required for calcium homeostasis and proper pollen tube tip growth in Petunia.</a> Planta. 245 (5), 909-926 (2017).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADK2+positively+modulates+abscisic+acid-induced+stomatal+closure+by+affecting+accumulation+of+H2O2,+Ca2++and+nitric+oxide+in+Arabidopsis+guard+cells.">NADK2 positively modulates abscisic acid-induced stomatal closure by affecting accumulation of H2O2, Ca2+ and nitric oxide in Arabidopsis guard cells.</a> Plant Science. 262, 81-90 (2017).</li><li>Niu, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnesium+availability+regulates+the+development+of+root+hairs+in+Arabidopsis+thaliana+(L.)+Heynh.">Magnesium availability regulates the development of root hairs in Arabidopsis thaliana (L.) Heynh.</a> Plant Cell and Environment. 37 (12), 2795-2813 (2014).</li><li>Qiu, L., Wang, Y., Qu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading+calcium+fluorescent+probes+into+protoplasts+to+detect+calcium+in+the+flesh+tissue+cells+of+Malus+domestica.">Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica.</a> Horticulture Research. 7, 91 (2020).</li><li>Boyer, J., Liu, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apple+phytochemicals+and+their+health+benefits.">Apple phytochemicals and their health benefits.</a> Nutrition Journal. 3 (1), 5 (2004).</li><li>Takahashi, A., Camacho, P., Lechleiter, J. D., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intracellular+calcium.">Measurement of intracellular calcium.</a> Physiological Reviews. 79 (4), 1089-1125 (1999).</li><li>Qu, H., Shang, Z., Zhang, S., Liu, L., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hyperpolarization-activated+calcium+channels+in+apical+pollen+tubes+of+Pyrus+pyrifolia.">Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia.</a> New Phytologist. 174 (3), 524-536 (2007).</li><li>Hadjantonakis, A. K., Pisano, E., Papaioannou, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tbx6+regulates+left/right+patterning+in+mouse+embryos+through+effects+on+nodal+cilia+and+perinodal+signaling.">Tbx6 regulates left/right patterning in mouse embryos through effects on nodal cilia and perinodal signaling.</a> PLoS One. 3 (6), 2511 (2008).</li><li>DeSimone, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+taste+receptor+cell+[Ca2+]i+modulate+chorda+tympani+responses+to+salty+and+sour+taste+stimuli.">Changes in taste receptor cell [Ca2+]i modulate chorda tympani responses to salty and sour taste stimuli.</a> Journal of Neurophysiology. 108 (12), 3206-3220 (2012).</li><li>Kao, J. P., Harootunian, A. T., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+generated+cytosolic+calcium+pulses+and+their+detection+by+fluo-3.">Photochemically generated cytosolic calcium pulses and their detection by fluo-3.</a> Journal of Biological Chemistry. 264 (14), 8179-8184 (1989).</li><li>Merritt, J. E., Mccarthy, S. A., Davies, M., Moores, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluo-3+to+measure+cytosolic+Ca2++in+platelets+and+neutrophils.+Loading+cells+with+the+dye,+calibration+of+traces,+measurements+in+the+presence+of+plasma,+and+buffering+of+cytosolic+Ca2.">Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2.</a> Biochemical Journal. 269 (2), 513-519 (1990).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+Ca+content+and+distribution+in+two+Gesneriaceae+species+reveals+distinctive+mechanisms+to+cope+with+high+rhizospheric+soluble+calcium.">A comparative study on Ca content and distribution in two Gesneriaceae species reveals distinctive mechanisms to cope with high rhizospheric soluble calcium.</a> Frontiers in Plant Science. 5 (5), 647 (2014).</li><li>Zhang, W., Rengel, Z., Kuo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+intracellular+Ca2++in+cells+of+intact+wheat+roots:+loading+of+acetoxymethyl+ester+of+Fluo-3+under+low+temperature.">Determination of intracellular Ca2+ in cells of intact wheat roots: loading of acetoxymethyl ester of Fluo-3 under low temperature.</a> Plant Journal. 15 (1), 147-151 (1998).</li><li>Qu, H., Jiang, X., Shi, Z., Liu, L., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+loading+ester+fluorescent+Ca2++and+pH+indicators+into+pollen+of+Pyrus+pyrifolia.">Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia.</a> Journal of Plant Research. 125 (1), 185-195 (2012).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+actin+filaments+induces+mitochondrial+Ca2++release+to+the+cytoplasm+and+[Ca2+]c+changes+in+Arabidopsis.+root+hairs.">Disruption of actin filaments induces mitochondrial Ca2+ release to the cytoplasm and [Ca2+]c changes in Arabidopsis. root hairs.</a> BMC Plant Biology. 10, 53 (2010).</li><li>Fujimori, T., Jencks, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lanthanum+inhibits+steady-state+turnover+of+the+sarcoplasmic+reticulum+calcium+ATPase+by+replacing+magnesium+as+the+catalytic+ion.">Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion.</a> Journal of Biological Chemistry. 265 (27), 16262-16270 (1990).</li></ol>

在这项研究中，通过酶水解获得了可行的原体。请注意，这种方法需要新鲜的苹果。本协议允许将大量原质从果浆中快速分离，用于研究。此方法的适用性不仅限于"富士"："杜南"和"蜂蜜脆"的苹果浆的原体也可以通过相同的协议提取（补充图S4）。酶溶解后的前端溶液包含细胞碎片，与以前的方法相比有所改进。作为一种必需的细胞材料，苹果浆原体可用于细胞蛋白表达技术、单细胞...

Ca2+在植物信号转导和代谢1、2中起着重要作用。此外，它调节水果质量特征3，4，包括硬度，糖含量，和易感性生理障碍在存储过程中5，6。细胞质Ca2+在信号转导中起着重要作用，调节植物生长发育。细胞钙质平衡的干扰可诱发苹果苦坑8个，梨9个棕色斑点病，西红柿脐腐烂10个，影响水果质量，造成严重经济损失3，11。钙成像具有足够的空间和时间分辨率，是观察活细胞12、13中Ca2+动力学的重要方法。
目前，活细胞细胞内钙成像主要有两种方法：一种采用化学小分子荧光探针14，另一种采用基因编码传感器（GECI）15、16。由于在果树中难以建立稳定的转基因系统和更长的果树发育，GECIS不适合果树Ca2+荧光成像。
小分子荧光探针，如Fluo-4/AM有一个特别的优势：他们的AM酯形式（细胞渗透乙酰酯衍生物）可以很容易地批量载入活细胞，而无需转染，这使得它灵活，快速，非细胞毒性17。Fluo-4/AM可以成功地装载到皮鲁斯皮里福利亚18和佩妮，19的花粉管，以及守卫细胞20和根毛的阿拉伯21。
目前，关于纸浆细胞钙荧光染色的报告很少。钙作为一种重要的矿物质元素，在苹果等树果的生长和质量控制中起着关键作用。苹果树被全球公认为重要的经济物种，苹果被认为是一种健康食品。在这项研究中，我们通过酶水解从苹果果浆中获得了可行的原质，然后将小分子荧光试剂装入细胞质中，以检测Ca2+。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10&#215; phosphate-buffered saline</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td>PBS (phosphate buffered solution) is a phosphate buffer solution, which can provide a relatively stable ionic environment and pH buffering capacity. It is a buffer salt solution often used in biology for molecular cloning and cell culture. The pH is 7.4.&#160;</td>
		</tr>
		<tr>
			<td>2-(N-morpholino)ethanesulfonic acid</td>
			<td>Solarbio</td>
			<td>M8010</td>
			<td>Biological buffer</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Solarbio</td>
			<td>C8370</td>
			<td>Calcium chloride dihydrate is a white or gray chemical, mostly in granular form.</td>
		</tr>
		<tr>
			<td>Cellulase R-10</td>
			<td>Yakult Honsha</td>
			<td>MX7352</td>
			<td>Degrade plant cell walls.</td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Solarbio</td>
			<td>S8090</td>
			<td>It has good moisturizing properties, prevents drying, and prevents sugar, salt, etc. from crystallizing.</td>
		</tr>
		<tr>
			<td>F-127</td>
			<td>Thermo Fisher Scientific</td>
			<td>P6867</td>
			<td>Pluronic F-127 is a non-ionic, surfactant polyol (molecular weight of approximately 12500 Daltons), which has been found to be beneficial to promote the dissolution of water-insoluble dyes and other materials in physiological media.&#160;</td>
		</tr>
		<tr>
			<td>FDA</td>
			<td>Thermo Fisher Scientific</td>
			<td>F1303</td>
			<td>FDA is a cell-permeant esterase substrate that can serve as a viability probe that measures both enzymatic activity, which is require to activate its fluorescence, and cell-membrane integrity, which is required for intracellular retention of their fluorescent product.&#160;</td>
		</tr>
		<tr>
			<td>Fluo-4/AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td>The green fluorescent calcium indicator Fluo-4/AM is an improved version of the calcium indicator Fluo-3/AM. The Fluo-4/AM loads faster and is brighter at the same concentration. It can be well excited with a 488 nm argon ion laser.</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Thermo Fisher</td>
			<td>EVOS Auto 2</td>
			<td>Observe the fluorescence image.</td>
		</tr>
		<tr>
			<td>Macerozyme R-10</td>
			<td>Yakult Honsha</td>
			<td>MX7351</td>
			<td>Degrade plant tissue to separate single cells.</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td>It is widely used in the preparation of buffers in biochemistry and molecular biology experiments.</td>
		</tr>
	</tbody>
</table>

staining the cytoplasmic ca2+ with fluo-4/am in apple pulp

细胞溶胶 Ca2+在植物开发中起着关键作用。钙成像是检测细胞质中 Ca2+动态变化的最通用方法。在这项研究中，我们通过酶水解获得了纸浆细胞的可行原体。分离的原质在37°C下用小分子荧光试剂（Fluo-4/AM）孵育30分钟。 荧光探针成功地染色了细胞溶胶Ca2+，但没有积聚在真空中。La3+， Ca2+通道阻滞剂， 降低细胞质荧光强度.这些结果表明，Fluo-4/AM可用于检测果肉中细胞溶胶 Ca2+的变化。总之，我们提出了一种方法，通过在果肉细胞的细胞质中加载小分子荧光钙试剂，有效地将原质从水果的肉细胞中分离出来，并检测Ca2+。

按照上述协议，我们使用酶方法从纸浆中获取可行的原质（图1）。有些原体有真空，而另一些没有。当Ca2+ 荧光指示器没有加载到原位时，原质没有荧光。当Flo-4/AM被加载到原质中时，细胞质，而不是真空，变成了荧光（图2）。这一结果表明，Fluo-4/AM成功地在细胞质中染色了Ca2+， 并且没有观察到24的隔膜化。原质被FD...

Watch this Scientific Journal Video about Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp at JoVE.com

Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp

苹果浆细胞的分离原体装有荧光钙试剂，以检测细胞质Ca2+ 浓度。

在苹果纸浆中用 Fluo-4/AM 染色细胞质 Ca2+

Cytosolic Ca2+ plays a key role in plant development. Calcium imaging is the most versatile method to detect dynamic changes in Ca2+ in the cytoplasm. In ...

钙作为一种重要的矿物质元素，在苹果等树果的生长和质量控制中起着关键作用，它能调节贮藏过程中的硬度、含糖量、储存期和生理紊乱。钙成像是检测细胞质中钙离子动态变化的最西方式。小分子荧光指标，如荧光-4/AM具有钙离子特异性选择性，可以通过酯化孵化进行非侵入性加载，这种孵化具有灵活性、快速性和非细胞毒性。该协议将引入一种将小分子化学荧光试剂荧光-4/AM加载到植物原质中的方法。前列提取将 0.5 毫升酶溶液加入 1.5 毫升离心机管中。选择一个健康成熟的苹果，然后切成10倍5倍1立方毫升大小的纸浆。将纸浆片放入酶溶液中，然后关闭管子。在28度孵化管一小时。晚上 70 点在黑暗中被棚屋。之后，洗涤纸浆片，吸气所有的酶溶液，然后加入0.5毫升的基本溶液。离心机以 300 g 的速度行驶 2 分钟。前体悬浮可以通过吸气底部溶液获得。小分子化学荧光试剂染色。准备氟-4/AM装载解决方案，以2毫摩尔氟-4/AM，20%乙基127和10倍磷酸盐缓冲盐水在一比一到二的比例。将氟-4/AM的1微升加载溶液添加到99微升前列座悬架。荧光染料的最终浓度为5微摩尔。混合溶液，然后关闭管子。同时，兰萨努姆铁钙或小分子化学荧光剂的独特性。加入一个微升或10微升以上的兰沙南铁，然后一个微升加载溶液的氟-4/AM到98微升原质悬架。兰萨努姆铁的最终浓度为100微摩尔。混合溶液，然后关闭管子。在37度下在黑暗中孵育30分钟。之后，离心机以300G的速度行驶两分钟。吸气到 17 微升溶液，并添加 17 微升基本溶液。将原位悬架在 37 度下孵化 30 分钟，以完全启动后，吸气 50 微升原质悬浮并滴入滑梯。在荧光显微镜下观察。选择 20 倍放大倍数和 GFP 通道进行观察。将亮度均匀调整为 0.5。这个建议方法的关键步骤是清洗原体中的污渍并孵育30分钟。在观察和孵化过程中，洗涤不足会导致荧光的过度背影，从而使细胞质的装载或探针更加充足。原体可生存性测定在37度孵育30分钟后，采取99微升原质悬浮。在 FD 溶液中加入 1 毫升，以达到原质悬架。通过上下管道多次混合溶液，然后关闭管子。在室温下在黑暗中停留五分钟。准备幻灯片，并使用 GFP 通道在荧光显微镜上观察幻灯片。我们向您展示结果如下图一。我们使用酶法从纸浆中获取原质。有些原体有真空，而另一些则没有。原质被FD染色了五分钟，在细胞质中显示荧光，表明高温37度不会影响活性或原质图二。原质在钙熨斗荧光指示器中不显示荧光，或者没有加载到它们中。比在荧光-4/AM被加载到前列座，细胞托普拉斯特，但没有一个真空成为荧光。有效阻断钙通道的兰萨努姆铁是细胞膜被添加到前列腺氟-4/AM加载。在100微摩尔的最终浓度，铁可以显著降低钙的荧光强度。这一结果进一步表明，氟-4/AM有效地停留在细胞质中的钙铁中，并显示出钙铁含量的动态变化。图三 使用IMH批准的加6.0软件荧光-4/AM染色，加上兰沙努姆铁，对荧光-4/AM结果进行了分析，显著降低了细胞质钙熨斗的荧光值。在这段视频中，所有的原质都是通过酶水解获得的。我们成功地将荧光探测器加载到37度的原质中。更过，该方法没有影响原质的生存能力。综上所述，这里详细描述了这种方法。苹果浆细胞细胞质钙离子的动态变化，为钙相关研究的进一步目的提供了10个相等的支持。

Research

JoVE Journal

Biology

Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp

Focal Ca2+ Transient Detection in Smooth Muscle

Focal Ca2+ Transient Detection in Smooth Muscle

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

科研

生物学

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

在体内的Ca 2 + -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Techniques for Imaging Ca2+ Signaling in Human Sperm

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...