Name: Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp (Video) | JoVE Journal
Uploaded: 2021-11-06T13:00:00.000+00:00
Duration: 8 min 5 s
Description: Learn to stain cytosolic Ca²⁺ in apple pulp protoplasts using Fluo-4/AM for plant cell imaging. This protocol enables noninvasive detection of calcium dynamics in fruit development research.

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5 minutes

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>

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</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><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

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...

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

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 ...

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Biology

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Learn to stain cytosolic Ca²⁺ in apple pulp protoplasts using Fluo-4/AM for plant cell imaging. This protocol enables noninvasive detection of calcium dynamics in fruit development research.

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

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