This&#160;work was supported by the Agricultural Variety Improvement Project of Shandong Province (2019LZGC007)&#160;and Fruit tree innovation team of Shandong modern agricultural industry&#160;technology system (SDAIT-06-05).

1. Protoplast extraction
<ol>
	<li>Prepare the basic solution: 20 mM CaCl2, 5 mM 2-(N-morpholino)ethanesulfonic acid, and 0.4 M D-sorbitol. 
	NOTE: The pH of the basic solution was adjusted to 5.8 with 0.1 M Tris buffer, filtered through 0.22 &#181;m water-soluble filters, and stored at 4 &#176;C.</li>
	<li>Prepare the enzymatic solution: Mix 0.3%(w/v) Macerozyme R-10 and 0.5%(w/v) cellulase R-10 with the basic solution.</li>
	<li>Add 0.5 mL of enzymatic solution into a 1.5 mL centrifuge tube. Pick a healthy and ripe apple. Then slice the pulp into 10 x 5 x 1 mm3 size (Figure 1A-1C).</li>
	<li>Place the apple fruit pulp pieces into a 1.5 mL centrifuge tube containing enzymatic solution and then close the tube (Figure 1D).</li>
	<li>Incubate the tube at 28 &#176;C for 1 h, shaking at 70 rpm/min in a shaker in the dark.</li>
	<li>Wash the pulp pieces. Aspirate all the enzymatic solution and then add 0.5 mL of the basic solution.</li>
	<li>Centrifuge at 300 x g for 2 min at room temperature.</li>
	<li>To obtain a protoplast suspension, aspirate the solution from the bottom of the centrifuge tube (Figure 1E).</li>
</ol>
2. Small-molecule calcium ion fluorescence staining
<ol>
	<li>Prepare the Fluo-4/AM loading solution with 2 mM Fluo-4/AM, 20% F-127, and 10x phosphate-buffered saline (PBS:80 mM Na2HPO4, 1.36 M NaCI, 20 mM KH2PO4, and 26 mM KCI) in a 1:1:2 ratio.</li>
	<li>Add 1 &#181;L of Fluo-4/AM loading solution to 99 &#181;L of the protoplast suspension present in the 1.5 mL centrifuge tubes. Ensure that the final concentration of the fluorescent dye is 5 &#181;M. Mix the solution and then close the tube.</li>
	<li>La3+ treatment: Prepare 100 &#181;M La3+ solution with 98 &#181;L of the protoplast suspension, 1 &#181;L of 10 mM La3+, and 1 &#181;L of Fluo-4/AM loading solution. Mix the solution and close the tube.</li>
	<li>Incubate for 30 min at 37 &#176;C in the dark.</li>
	<li>Wash the protoplasts by centrifugation at 300 x g for 2 min at room temperature. Aspirate 70 &#181;L of the solution and add 70 &#181;L of the basic solution.</li>
	<li>Incubate the protoplast suspension at 37 &#176;C for 30 min to completely de-esterify.</li>
	<li>Aspirate 15 &#181;L of the protoplast suspension and drip onto a slide.</li>
	<li>Observe under a fluorescence microscope (Supplementary Figure S1). 
	NOTE: Use a color camera with high sensitivity, i.e., 3.2 MP (2048 x 1536) CMOS sensor with 3.45 &#181;m pixel resolution.</li>
	<li>Select the GFP channel for imaging (20x). Set the brightness to 0.5. 
	&#8203;NOTE:Illumination is adjustable-intensity LED light cubes with an integrated hard-coated filter set. The excitation wavelength of Fluo-4/AM is 490 nm.</li>
</ol>
3. Protoplast viability assay
<ol>
	<li>Prepare the Fluorescein diacetate (FDA) stock solution: Dissolve FDA in acetone until the final concentration is 1 mg/mL.</li>
	<li>Prepare the FDA working solution with 1 &#181;L of the stock solution and 99 &#181;L of acetone.</li>
	<li>Add 1 &#181;L of FDA working solution to 99 &#181;L of protoplast suspension. Mix the solution by pipetting up and down and then close the tube. 
	NOTE: The final concentration of the FDA is 100 &#181;g/L.</li>
	<li>Stain at room temperature for 5 min in the dark.</li>
	<li>Prepare the slides and observe under a fluorescence microscope.</li>
	<li>Select the GFP channel for imaging (Figure 1F).</li>
</ol>
4. Image analysis
<ol>
	<li>Analyze the acquired images using image analysis and spreadsheet software (e.g., Image-Pro Plus and Excel 2010).</li>
	<li>Select two vertical diameters from the protoplasts to calculate fluorescence intensity (Supplementary Figure S2). Measure the fluorescence intensity of all protoplasts under different treatments was measured. For final processing, use photo editing software.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Perform statistical analysis using statistical software (Supplementary Figure S3). Data are presented in Mean &#177; SD. The student&#39;s t-test was used to analyze the differences between the experimental groups.</li>
</ol>

<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>

The authors declare that they have no conflicts of interest with the contents of this article.

In this study, viable protoplasts were obtained by enzymatic hydrolysis. Note that this method requires fresh apples. The present protocol allows for the rapid isolation of a large number of protoplasts from fruit pulp for use in research studies. The applicability of this method is not limited to &#39;Fuji&#39;; the protoplasts of the apple pulp of &#39;Dounan&#39; and &#39;Honey Crisp&#39; can also be extracted through the same protocol (Supplementary Figure S4). The protoplast solution after enzymolys...

Ca2+ plays an important role in plant signal transduction and metabolism1,2. Further, it regulates fruit quality traits3,4, including hardness, sugar content, and susceptibility to physiological disorders during storage5,6. Cytoplasmic Ca2+ plays an important role in signal transduction and regulates plant growth and development7. Disturbance of cellular calcium homeostasis can induce bitter pit in apples8, brown spot disease in pears9, and umbilical rot in tomatoes10, affecting fruit quality and causing severe economic losses3,11. Calcium imaging has sufficient spatial and temporal resolution and is an important method for observing Ca2+ dynamics in living cells12,13.
At present, there are two main methods for intracellular calcium imaging in live cells: one employs chemical small-molecular fluorescent probes14, and the other is the gene encoding sensor (GECI)15,16. Given the difficulty of establishing a stable transgenic system in fruit trees and longer fruit development, GECIS is unsuitable for fruit Ca2+ fluorescence imaging.
Small-molecule fluorescent probes such as Fluo-4/AM have a particular advantage: their AM ester form (cell-permeable acetoxymethyl ester derivative) can be readily bulk-loaded into living cells without the need for transfection, which makes it flexible, rapid, and non-cytotoxic17. Fluo-4/AM could successfully be loaded into the pollen tube of Pyrus pyrifolia18 and Petunia,19 as well as into guard cells20 and root hair of Arabidopsis21.
At present, there are few reports on the calcium fluorescence staining of pulp cells22. As an important mineral element, calcium plays a key role in the growth and quality control of tree fruits such as apples. Apple trees are globally recognized as an important economic species, and apples are considered a healthy food23. In this study, we obtained viable protoplasts from apple fruit pulp through enzymatic hydrolysis and then loaded small-molecule fluorescent reagents into the cytoplasm to detect 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

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 this study, we obtained viable protoplasts of pulp cells by enzymatic hydrolysis. Isolated protoplasts were incubated with the small-molecule fluorescent reagent (Fluo-4/AM) for 30 min at 37 &#176;C. The fluorescent probes successfully stained cytosolic Ca2+ but did not accumulate in vacuoles. La3+, a Ca2+ channel blocker, decreased cytoplasmic fluorescence intensity. These results suggest that Fluo-4/AM can be used to detect changes in cytosolic Ca2+ in the fruit flesh. In summary, we present a method to effectively isolate protoplasts from flesh cells of the fruit and detect Ca2+ by loading a small-molecule calcium fluorescent reagent in the cytoplasm of pulp cells.

Following the protocol described above, we used the enzymatic method to obtain viable protoplasts from the pulp (Figure 1). Some protoplasts had vacuoles, while others did not. While the protoplasts exhibited no fluorescence when the Ca2+ fluorescent indicator was not loaded into them. When Fluo-4/AM was loaded into the protoplasts, the cytoplasm, but not the vacuole, became fluorescent (Figure 2). This result indicated that Fluo-4/AM successfully sta...

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

Isolated protoplasts of apple pulp cells were loaded with a calcium fluorescent reagent to detect cytoplasmic Ca2+ concentration.

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

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 ...

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

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JoVE Journal

Biology

4.1K Views.  Qingdao Agricultural University. Isolated protoplasts of apple pulp cells were loaded with a calcium fluorescent reagent to detect cytoplasmic Ca2+ concentration.

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

Focal Ca2+ Transient Detection in Smooth Muscle

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+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

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+ ...

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

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 measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

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

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

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

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

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

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

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

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

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

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

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

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

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

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

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

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

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

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 (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

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

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

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

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

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