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 enzymolysis contains cell debris, which was somewhat improved compared to previous methods. As an essential cell material, apple pulp protoplasts can be used for cell protein expression technology, single-cell sequencing, and other research.
There are many methods regarding chemical fluorescent reagent staining in plant cells30. For example, Fluo-3/AM was loaded at a low temperature (4 &#176;C), such that it would enter the root tip cells31. Fluo-3/AM can also be loaded at a high temperature (37 &#176;C) into the pollen tube32. Fluo-4/AM has successfully loaded into the pollen tube of Pyrus pyrifolia (25 &#176;C for 15 min)18 and the root hair of Arabidopsis (4 &#176;C for 30 min)33. However, these methods are not suitable for staining apple pulp protoplasts. In this study, we successfully loaded fluorescent probes into protoplasts at a high temperature (37 &#176;C). In addition, the present method is simple, fast, accurate, and does not affect protoplast vitality. A critical step in the proposed method is to wash the stained protoplasts and incubate them for 30 min. After staining, the protoplasts should be washed. The washing will reduce the background fluorescence during observation so that the fluorescence intensity of the protoplasts can be counted more accurately. Incubate for 30 min to allow more probes to be loaded into the protoplasts. Note that it is essential to avoid light exposure during the loading.
La3+ is an important tool for studying Ca2+. It binds to the Mg2+ catalytic site on the cytoplasmic membrane Ca2+-ATPase, thereby inhibiting the steady-state turnover of Ca2+-ATPase and blocking the Ca2+ transmembrane functioning34. La3+ treatment reduced cytoplasmic Ca2+, and Fluo-4/AM could reflect this change, excluding the interference of other divalent cations and verifying the feasibility of chemical fluorescence reagents.
Although this loading method offers more advantages than other methods, it still needs to be further improved in transforming data results. Here, we estimated the relative cytoplasmic Ca2+ content by detecting the fluorescence value of protoplasts, which is qualitative data rather than quantitative. Therefore, it is particularly important to translate the staining results into specific cytoplasmic Ca2+ content in future studies, which could provide a research basis for analyzing fruit Ca2+ signaling.
In summary, the method described herein can detect cytoplasmic Ca2+ in apple pulp cells, thereby providing technical support for the related studies of fruit pulp-cell calcium.

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><tbody><tr><td>10&amp;times; 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.&amp;nbsp;</td></tr><tr><td>2-(N-morpholino)ethanesulfonic acid</td><td>Solarbio</td><td>M8010</td><td>Biological buffer</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</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.&amp;nbsp;</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.&amp;nbsp;</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 stained Ca2+ in the cytoplasm and that no compartmentalization was observed24. Protoplasts were stained with FDA for 5 min and showed cytoplasmic fluorescence. This indicated that high temperature (37 &#176;C) does not affect protoplast viability.
La3+, a blocking agent of the Ca2+ channel25, was added when Fluo-4/AM was loaded into protoplasts. At 100 &#181;M, La3+ decreased calcium fluorescence intensity (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62526/62526fig01.jpg" /> 
Figure 1: Protoplasts obtained by enzymatic hydrolysis. (A) A piece was cut from a mature apple. (B) Protoplasts were extracted from pulp pieces. (C) The pulp was cut into 10 &#215; 5 &#215; 1 mm3 pieces. (D) Pulp pieces were placed in centrifuge tubes containing 0.5 mL enzyme solution. (E) Protoplasts. Arrowheads point to the protoplast, and arrows indicate the vacuole in the protoplast. (F) Protoplasts were stained with FDA for 5 min at room temperature. (This figure has been modified from reference 22 [Horticulture research: Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica]. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62526/62526fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62526/62526fig02.jpg" /> 
Figure 2: Loading Fluo-4/AM and La3+ into the protoplasts. (A) Control: Intact protoplast without any loaded fluorescent probe. (B) Protoplasts loaded with Fluo-4/AM. (C) La3+ was added when the protoplasts were loaded with Fluo-4/AM. The final concentration of La3+ was 100 &#181;M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62526/62526fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62526/62526fig03.jpg" /> 
Figure 3: Statistical analysis of fluorescence intensity in the protoplasts. *** Indicate significant difference as per Student&#180;s t-test (P &#60;0.001). Vertical bars indicate &#177; SD. Each data point represents the mean of 20 protoplasts. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62526/62526fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Fluorescence microscope. (A) The overall appearance of the fluorescence microscope. (B) Settings page. Select 20x objective, GFP channel, and uniformly adjust the brightness to 0.5. (C) Fluorescence excitation region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62526/Supplementary Fig S1.docx" target="_blank">Please click here to download this File.</a>
Supplementary Figure S2: Steps used to calculate the Ca2+ fluorescence intensity at protoplast of fruit cell. Fluorescence intensity units used in this study are based on previous reports26,27,28,29. The calculation process is as follows:&#160;(A)&#160;Open the protoplast fluorescence image in the software. Click on the Measure tool. From the dropdown menu select Profile Line. (B) Select Circle in the Line Profile window. Using this draw an ellipse at the protoplast. (C)&#160;In the Line Profile window now select File&#160;| Export data (D) Ensure that the blank spreadsheet is opened and import data by clicking Data Export. Use the &#39;average&#39; function to calculate the average fluorescence intensity. Each treatment was repeated three times with more than 20 protoplasts each. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62526/Supplementary Fig S2.docx" target="_blank">Please click here to download this File.</a>
Supplementary Figure S3: Data statistics were performed using statistical analysis software. Paste the data into the table and click Analyze for data analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62526/Supplementary Fig S3.docx" target="_blank">Please click here to download this File.</a>
Supplementary Figure S4: Extraction of protoplasts from the pulp of other varieties of apples. (A) Dounan. (B) Honey Crisp. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62526/Supplementary Fig S4.docx" target="_blank">Please click here to download this File.</a>

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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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4.3K 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

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neuroscience

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Source: Cen, W., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.</a> J. Vis. Exp. (2022)
In this video, we describe an in vitro osteoclastic resorption assay on calcium phosphate-coated cell culture plates cultured with human peripheral blood mononuclear cell-derived osteoclasts. The osteoclasts form a characteristic actin ring on the calcium phosphate-coated matrix of the cell culture plate and degrade the matrix within the actin ring region to release calcium and phosphate ions, resulting in the formation of distinct resorption pits within the degraded matrix.

Test resorpcyjny: technika in vitro do ilościowego określania aktywności resorpcyjnej dojrzałych osteoklastów przy użyciu płytek hodowlanych pokrytych fosforanem wapnia

Source: Cen, W., et al. A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro. J. Vis. Exp. (2022)
In this video, we ...

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Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye

Source: Perrenoud, L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64526">Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry.</a>&#160;J. Vis. Exp. (2022)
This video analyzes T-cell receptor-induced calcium influx in T-cells. Anti-CD3 antibodies activate the T-cell signaling pathways, leading to an increase in intracellular calcium ions that bind with Indo-1 dye. In flow cytometry, a spectrum shift from the calcium-free state to the calcium-bound state&#160; correlates with T-cell receptor-induced calcium influx.

Ocena napływu wapnia indukowanego przez receptor limfocytów T za pomocą barwnika wskaźnikowego wapnia

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

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