Name: Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels (Video) | JoVE Journal
Uploaded: 2024-12-13T14:20:00.000+00:00
Duration: 7 min 17 s
Description: Learn single-cell calcium imaging using Fura-2/AM dye to monitor TRPV1 channel activation and thermal responses in keratinocytes and HEK293T cells. This method enables real-time quantitative analysis of calcium signaling for pain and temperature sensation research.

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5 minutes

Acknowledgment is given to Bailong Xiao from Tsinghua University for sharing the single-cell Ca2+ imaging system and the temperature control operating system, as well as for the support and assistance in this project. This research was funded by the National Natural Science Foundation of China (32000705), the Young Elite Scientists Sponsorship Program by the China Association of Chinese Medicine (CACM-(2021&#8211;QNRC2&#8211;B11)), Fundamental Research Funds for the Central Universities (2020&#8211;JYB&#8211;XJSJJ&#8211;026), (2024-JYB-KYPT-06).

The experimental methods were approved by and followed the IACUC guidelines of Tsinghua University and Beijing University of Chinese Medicine. This protocol introduces single-cell Ca2+ imaging methods for various cell types, including primary keratinocytes isolated from the skin of several newborn mice (within three days of birth, with sex-randomized littermates, C57BL/6 mice). Details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Cell preparation
NOTE: Primary cells, cell lines with endogenous target genes, or those transfected with overexpressed plasmids are all suitable for single-cell Ca2+ imaging. The plasmids used in this study were obtained from Professor Xiao Bailong&#39;s laboratory at Tsinghua University. These plasmids were constructed by incorporating sequences of GFP fluorescent protein with human STIM1, DsRed fluorescent protein with human Orai1, mRuby fluorescent protein with rabbit TRPV1, as well as the red fluorescent protein mCherry into phage plasmid vectors10.
<ol>
	<li>Prepare sterile glass slides with an 8 mm diameter in a 24-well plate. Add 500 &#956;L of poly-D-lysine (PDL) buffer (50 &#956;g/mL in DPBS) to each well.</li>
	<li>Incubate the slides at 37 &#176;C for 1 h to allow coating, then discard the coating solution using a pipette.</li>
	<li>Wash the slides once with DPBS and set them aside for later use.</li>
	<li>Culture primary cells and cell lines separately according to their specific cultivation methods10.</li>
	<li>Seed cells onto the prepared 24-well plate at a density of approximately 1.5 x 105 cells per well. Use the cells for Ca2+ imaging once they have adhered to the coverslip.</li>
	<li>For cells overexpressing plasmids, transfect10,15 the target plasmid (1 &#956;g/well) using Lipofectamine 2000 (or Lipofectamine 3000) transfection reagent at a 1:1 ratio, and incubate in a cell culture incubator for about 24 h. 
	NOTE: A longer incubation time may be necessary for larger expressed proteins.</li>
</ol>
2. Preparation of Fura-2/AM working solution
<ol>
	<li>Add 50 &#956;L of dimethyl sulfoxide (DMSO) to a tube containing 50 &#956;g of Fura-2/AM powder and mix well to prepare a 1 &#956;g/&#956;L stock solution of Fura-2/AM.</li>
	<li>Mix the Fura-2/AM stock solution and Pluronic F-127 into Hank&#39;s buffer containing 1.3 mM Ca2+. 
	NOTE: The final concentration of Fura-2/AM and Pluronic F-127 in the working solution is 2.5 &#956;g/mL. The Hank&#39;s buffer is prepared by adding 10 mM HEPES to 1x HBSS buffer.</li>
	<li>Use aluminum foil to protect the Fura-2/AM working solution from light.</li>
</ol>
3. Cell pretreatment for single-cell Ca2+ imaging
<ol>
	<li>Transfer the glass slides with cells to a new 24-well plate containing Hank&#39;s buffer for washing.</li>
	<li>Discard the buffer using a pipette and add 500 &#956;L of Fura-2/AM working buffer to each well.</li>
	<li>Incubate at room temperature in the dark for 30 min to allow probe loading.</li>
	<li>Remove the Fura-2/AM working buffer and wash the cells three times with Hank&#39;s buffer to eliminate excess Fura-2/AM. The cells are now ready for use.</li>
</ol>
4. Starting the Ca2+ imaging system
NOTE: In this study, a fluorescence microscope is used for Ca2+ imaging.
<ol>
	<li>Start the following components in sequence: DG4 light (xenon lamp), camera, white-light source, microscope stage controller, microscope, computer, and fluorescence imaging software. 
	NOTE: If detecting the activation of ion channels by temperature, also turn on the following components: perfusion system, heating system, temperature controller, and liquid circulation heating/cooling device.</li>
</ol>
5. Cellular Ca2+ response procedure
<ol>
	<li>Open the fluorescence imaging software (see Table of Materials).
	<ol>
		<li>Choose Protocol, then select File, followed by Load Protocol. Select the protocol and click on OK.</li>
		<li>Configure the experiment (on the menu list).</li>
		<li>Select New Experiment.</li>
	</ol>
	</li>
	<li>Mount the perfusion chamber on the microscope. 
	NOTE: Always lower the objective fully when mounting or removing chambers using the rough focus knob to prevent damage to the objective.</li>
	<li>Remove the Fura-2/AM treated cell slides and place them in the chamber containing Hank&#39;s buffer.</li>
	<li>Start the perfusion system.</li>
	<li>Select the 20x DIC objective and adjust the focus under white light.</li>
	<li>Click on Cfg Exp on the taskbar.
	<ol>
		<li>Select the desired fluors for imaging.</li>
		<li>Determine the acquisition frequency and display settings on the screen (Acquire: check for 340, 380, GFP; Acquire Interval: 1 s; Save Interval: 1 s).</li>
	</ol>
	</li>
	<li>Focus
	<ol>
		<li>On the experiment control panel, click on the Focus button.</li>
		<li>Adjust the acquisition time (usually 100 ms) and gain as needed, then &#34;save for this wave.&#34; 
		NOTE: For UV light, use gain instead of exposure time.</li>
		<li>Choose the desired wavelength for focusing (e.g., 380) and click on Start Focusing.</li>
		<li>Switch the view from the binoculars to the computer.</li>
		<li>Ensure that a faint greenish color is visible through the binoculars.</li>
		<li>Focus on the cells, using rough focus first to get the objective close, then fine focus. 
		NOTE: The microscope will beep if it gets too close to the stage. If this happens, lower the objective, realign the plate on the stage, and focus again. Minimize time spent focusing to reduce laser-induced cell damage.</li>
		<li>Check the fluorescence intensity of the cells during the focusing process.</li>
		<li>Adjust the fluorescence intensity of the cells by modifying the exposure time and gain. 
		NOTE: The exposure time and gain for 340 and 380 must be consistent.</li>
		<li>Once a good focus is achieved, press the button on the microscope to switch the view to the computer.</li>
		<li>Refocus as needed to obtain the sharpest image on the computer, then click on Stop Focusing.</li>
	</ol>
	</li>
	<li>Alternate procedure for finding GFP positive cells
	<ol>
		<li>Use the 340/380 procedure first to achieve a good focus on the cells.</li>
		<li>Select FITC and then click on Start Focusing.</li>
		<li>Use the stage controller to find GFP-positive cells.</li>
		<li>Switch the view to the computer and then click on Stop Focusing.</li>
	</ol>
	</li>
	<li>Region selection
	<ol>
		<li>Click on the Region button in the menu bar.</li>
		<li>Select the illumination type of choice (340/380/FITC/TRITC). FITC and TRITC filters are selected for GFP and mCherry/DsRed/mRuby, respectively, for cells overexpressing the targeted plasmids; otherwise, select Fura-2. 
		NOTE: Avoid selecting cells that are in poor condition or dead, such as those that are obviously rounded or have overexposed fluorescent proteins.</li>
		<li>Click on Acquire Images, then OK.</li>
		<li>A new window will appear; select cells by clicking on the oval tool and then clicking over the cell.</li>
		<li>Select the desired cells under the fluorescence carried by the target protein (FITC or TRITC). Select the control cells that do not express the target protein under the 380 nm condition.</li>
		<li>Undo a region by right-clicking on the circle and then selecting Delete Region.</li>
		<li>Select a background sample as the last region and record its ID number.</li>
		<li>Click on Save and then Done.</li>
	</ol>
	</li>
	<li>Background subtraction
	<ol>
		<li>Click on the menu button References.</li>
		<li>Indicate the number of the background region. 
		NOTE: If the background was the last region selected, entering a very high number will automatically switch to the last region picked.</li>
		<li>Check the box Subtract References and then click on OK.</li>
	</ol>
	</li>
	<li>Log data
	<ol>
		<li>Click on the Log Data button on the experiment control panel. 
		NOTE: Images are saved only if there is a desire to replay the experiment later; normally, just checking the data box is sufficient.</li>
		<li>Ensure that a prompt appears asking for the preferred data file type; selecting .xlsx format is recommended.</li>
		<li>An &#34;.xlsx&#34;&#160;type worksheet will open. Minimizing the worksheet will prevent it from taking up screen space.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>In the control panel&#39;s Time Lapse section, set the data acquisition interval to 1 s. 
		NOTE: This can be adjusted according to actual needs.</li>
		<li>Click on Zero Clock and Acquire on the experiment control panel to start the experiment. Baselines will be visible on a graph indicating each of the selected cell regions.</li>
		<li>Perform a series of treatments on the cells according to the experimental requirements.</li>
		<li>To observe the temperature response of the cells, heat the buffer in the perfusion system to an appropriate temperature using a temperature controller.</li>
		<li>To observe the effects of drugs on cells, perfuse or manually add drug-containing buffer and record the cell changes.</li>
		<li>After data acquisition is complete, click on Pause. 
		NOTE: Data acquisition can be paused, and the clock can be reset at any time during the experiment.</li>
		<li>Save and analyze the data.</li>
	</ol>
	</li>
	<li>Click on File and Close Experiment to end the experiment, and select No on the dialog box to save the protocol.</li>
	<li>Open a new experiment by clicking on New on the menu, and repeat the process.</li>
	<li>Shut down
	<ol>
		<li>Close the software and transfer the data from the computer.</li>
		<li>Reverse the start-up procedure.</li>
		<li>Log the hours on the sign-up sheet and clean up any mess.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Represent the intracellular Ca2+ concentration by the Fura-2 340/380 ratio or convert to the corresponding Ca2+ concentration.</li>
	</ol>
	</li>
</ol>

Single Cell Calcium Imaging for Channel Activation Studies

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<li>Prakriya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orai1+is+an+essential+pore+subunit+of+the+crac+channel%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Orai1 is an essential pore subunit of the crac channel.</a> Nature. 443 (7108), 230-233 (2006).</li>
<li>Caterina, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+capsaicin+receptor%3A+A+heat-activated+ion+channel+in+the+pain+pathway%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The capsaicin receptor: A heat-activated ion channel in the pain pathway.</a> Nature. 389 (6653), 816-824 (1997).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stim1+thermosensitivity+defines+the+optimal+preference+temperature+for+warm+sensation+in+mice%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">Stim1 thermosensitivity defines the optimal preference temperature for warm sensation in mice.</a> Cell Res. 29 (2), 95-109 (2019).</li>
<li>Bharate, S. S., Bharate, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+thermoreceptor+trpm8+by+cooling+compounds%5BTitle%5D)+AND+%22ACS+Chem+Neurosci%22%5BJournal%5D)">Modulation of thermoreceptor trpm8 by cooling compounds.</a> ACS Chem Neurosci. 3 (4), 248-267 (2012).</li>
<li>Coste, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Piezo+proteins+are+pore-forming+subunits+of+mechanically+activated+channels%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Piezo proteins are pore-forming subunits of mechanically activated channels.</a> Nature. 483 (7388), 176-181 (2012).</li>
<li>Behera, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analyses+of+Ca2%2B+dynamics+using+a+ubiquitin-10+promoter-driven+yellow+cameleon+3.6+indicator+reveal+reliable+transgene+expression+and+differences+in+cytoplasmic+Ca2%2B+responses+in+arabidopsis+and+rice+%28oryza+sativa%29+roots%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">Analyses of Ca2+ dynamics using a ubiquitin-10 promoter-driven yellow cameleon 3.6 indicator reveal reliable transgene expression and differences in cytoplasmic Ca2+ responses in arabidopsis and rice (oryza sativa) roots.</a> New Phytol. 206 (2), 751-760 (2015).</li>
<li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high+signal-to-noise+ca%282%2B%29+probe+composed+of+a+single+green+fluorescent+protein%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">A high signal-to-noise ca(2+) probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19 (2), 137-141 (2001).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanism+analysis+of+stim1+thermal+sensation%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Molecular mechanism analysis of stim1 thermal sensation.</a> Cells. 12 (22), 2613(2023).</li>
<li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATF6+safeguards+organelle+homeostasis+and+cellular+aging+in+human+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Cell+Discov%22%5BJournal%5D)">ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells.</a> Cell Discov. 4, 2(2018).</li>
<li>Jiang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanosensitive+piezo1+channel+mediates+heart+mechano-chemo+transduction%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">The mechanosensitive piezo1 channel mediates heart mechano-chemo transduction.</a> Nat Commun. 12 (1), 869(2021).</li>
<li>Tao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+Orai1-mediated+store-operated+Ca%282%2B%29+channel%2Fcalpain+signaling+contributes+to+high+glucose-induced+podocyte+injury%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Enhanced Orai1-mediated store-operated Ca(2+) channel/calpain signaling contributes to high glucose-induced podocyte injury.</a> J Biol Chem. 298 (6), 101990(2022).</li>
<li>Guo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+er+ion+homeostasis+maintained+by+an+er+anion+channel+clcc1+contributes+to+als-like+pathologies%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">Disruption of er ion homeostasis maintained by an er anion channel clcc1 contributes to als-like pathologies.</a> Cell Res. 33 (7), 497-515 (2023).</li>
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The authors declare that they have nothing to disclose.

The application of single-cell Ca2+ imaging systems is extensive, enabling the study of Ca2+ signals in various cell types, including keratinocytes, stem cells16, liver cells, heart cells17, podocytes18, immune cells, and cell lines overexpressing target proteins10,19. This technique measures changes and absolute values of cellular Ca2+ concentrations and plays a crucial role in investigating Ca2+-related ion channels, making it one of the essential instruments in ion channel research laboratories.
As research progresses, the significance of Ca2+ signal-related ion channels has gained increasing recognition among traditional Chinese medicine researchers. They aim to decode the core issues of traditional Chinese medicine through the lens of ion channels. For instance, the hot and cold properties of Chinese medicine inform the principle of &#34;treating heat with cold and treating cold with heat.&#34; Many temperature-sensitive ion channels that mediate Ca2+ signals are closely related to this principle. Furthermore, numerous Chinese medicines exert therapeutic effects through anti-inflammatory actions, with multiple Ca2+-related ion channels playing significant roles in the regulation of inflammation20.
It is essential to study the effects of important components in traditional Chinese medicine on ion channel characteristics to reveal the pharmacological properties of these medicines at the molecular level. Currently, researchers in traditional Chinese medicine primarily focus on detecting the expression of relevant ion channels. However, there is still relatively limited real-time monitoring regarding the effects of traditional Chinese medicine on Ca2+-related ion channels, and many underlying mechanisms remain unclear.
This research group has been dedicated to the research of Ca2+-related ion channels, such as STIM1/Orai1, STIM1/Orai3, TRPA110, and TRPV1, and is well-versed in the application and detailed usage of single-cell Ca2+ imaging systems. This study demonstrates the specific usage of single-cell Ca2+ imaging for different research purposes, aiming to provide readers with a comprehensive understanding of its extensive applications and detailed usage. Compared to previously published related studies21, this research offers a new perspective and methodology for the application of single-cell Ca2+ imaging from various angles and in conjunction with other systems. For example, primary cells or cell lines with endogenous expression of target genes can be directly loaded with the Fura-2 probe to monitor changes in intracellular Ca2+ signals in real-time under various stimulating factors using the single-cell Ca2+ imaging system. Additionally, overexpression of plasmids containing fluorescent proteins in cell lines can be performed. Initially, the cells are loaded with the Fura-2 probe, as described earlier. Next, cells with successful expression of the target protein are selected based on FITC or TRITC signals, which are then highlighted for subsequent real-time monitoring of Fura-2 Ca2+ signals.
Single-cell Ca2+ imaging is a detection system that can be integrated with various other experimental setups according to the experimental objectives. For example, if the response of temperature-sensitive ion channels to temperature changes needs to be detected, the system can be combined with a temperature control operation system and a perfusion device to simultaneously monitor changes in intracellular Ca2+ signals by controlling the temperature of the extracellular buffer solution19. This approach confirms the response of the relevant ion channels to temperature. Moreover, single-cell Ca2+ imaging can also be coupled with relevant drug delivery devices or other cell stimulation devices for related experiments.
In fact, the single-cell Ca2+ imaging system not only allows for real-time monitoring of the 340/380 ratio but also the values of FITC or TRITC through specific settings. Furthermore, single-cell calcium imaging technology is applicable for detecting changes in calcium ion concentration not only in the cytoplasm but also in the endoplasmic reticulum (ER), demonstrating its wide-ranging applications19. The data obtained by this system are uniformly saved in an &#34;.xlsx&#34;&#160;format, which can be quickly analyzed to obtain results. The change curve facilitates a quick understanding of the activation status and characteristics of cells, establishing a convenient Ca2+ signal monitoring system. It is anticipated that this research report will effectively promote the widespread application of the single-cell Ca2+ imaging system in the field of traditional Chinese medicine research and contribute to decoding traditional Chinese medicine.
However, the application of single-cell calcium imaging technology does have certain limitations, such as the inability to achieve high-throughput screening. For example, in some experiments, when it is necessary to screen for agonists or antagonists of a specific channel, calcium imaging requires screening one by one, which is very time-consuming. In such cases, this technique may need to be combined with other technologies, such as FLIPR22, to perform high-throughput screening before further validating the results with single-cell calcium imaging. Additionally, the images obtained from single-cell calcium imaging are generated from conventional fluorescence microscopy, which has a lower resolution compared to confocal microscopy15. Therefore, effectively combining single-cell calcium imaging technology with other techniques to leverage their respective advantages is an important approach to addressing challenges in this field.

Ca2+ plays a crucial role in cellular signal transduction, regulating various cellular functions such as muscle contraction1, nerve conduction2, secretion3, and gene expression4, thereby influencing multiple physiological processes. Abnormal Ca2+ concentrations can lead to diseases such as arrhythmias5, coagulation disorders6, and hormonal imbalances7. Therefore, studying the mechanisms of intracellular Ca2+ concentration changes is of paramount importance.
Various ion channels are involved in the regulation of Ca2+ concentration in cells, including highly Ca2+-selective calcium release-activated calcium (CRAC) channels8 and non-selective cation channels of the TRP family9. These ion channels can be activated by stimuli such as temperature10, compounds, and active ingredients found in traditional Chinese medicine11, playing a crucial role in various Ca2+-related physiological processes.
Effective monitoring of intracellular Ca2+ concentration changes is essential for studying Ca2+-related ion channels, particularly in the field of traditional Chinese medicine, where calcium signaling regulation plays a central role in many therapeutic approaches. Currently, the primary methods for measuring intracellular Ca2+ can be categorized into two types: electrical and optical measurements. The electrical measurement approach uses the patch-clamp technique to assess changes in cell membrane potential due to Ca2+ influx12.
In optical measurement, fluorescent probes specifically bind to Ca2+, allowing researchers to track changes in cellular fluorescence intensity. Common optical methods include fluorescent protein-based and fluorescent dye-based techniques. In fluorescent protein-based methods, researchers can overexpress Ca2+-sensitive fluorescent proteins like Cameleon13 and GCaMP14 in cells and monitor fluorescence signal changes using fluorescence microscopy or flow cytometry to observe shifts in cytoplasmic Ca2+ concentrations. Additionally, researchers can overexpress these proteins in mice and use two-photon fluorescence microscopy for real-time in vivo or tissue-level monitoring of intracellular Ca2+ concentrations, providing high resolution and deep tissue penetration10.
For fluorescent dye-based methods, commonly used Ca2+ probes include Fluo-3/AM, Fluo-4/AM, and Fura-2/AM10. Researchers incubate cells in a solution containing these fluorescent probes, which cross the cell membrane and are cleaved by intracellular esterases to form active compounds (e.g., Fluo-3, Fluo-4, and Fura-2) that remain within the cell. These probes exhibit minimal fluorescence in their free ligand form but emit strong fluorescence when bound to intracellular Ca2+, thereby indicating changes in cytoplasmic Ca2+ concentrations. Compared to other fluorescent proteins and dyes, Fura-2 is typically excited at 340 nm and 380 nm wavelengths. When bound to intracellular free Ca2+, Fura-2 undergoes an absorption shift, moving the excitation wavelength peak from 380 nm to 340 nm, while the emission peak near 510 nm remains unchanged. There is a quantitative relationship between fluorescence intensity and bound Ca2+ concentration, allowing calculation of intracellular Ca2+ concentration by measuring the fluorescence intensity ratio at these two excitation wavelengths. Ratio measurements reduce the effects of photobleaching, fluorescent probe leakage, uneven loading, and differences in cell thickness, yielding more reliable and reproducible results (Figure 1).
Single-cell Ca2+ imaging systems primarily utilize microscopy techniques and the Ca2+ indicator Fura-2/AM to detect intracellular Ca2+ concentrations. These systems comprise a fluorescence microscope, a Ca2+ imaging light source, and fluorescence imaging software, enabling real-time, quantitative monitoring of Ca2+ changes in the cytoplasm of multiple cells simultaneously (up to 50 cells per field of view). Results are saved in &#34;.xlsx&#34;&#160;format for subsequent analysis. The system offers a rapid analysis speed (approximately 1 min for analyzing a group of cells within one field of view) and generates intuitive change curves, significantly enhancing detection efficiency. Single-cell Ca2+ imaging is an essential technical approach for studying Ca2+-related channels and has considerable value in ion channel-related biomedical research. Its application in single-cell calcium imaging technology is expected to greatly advance research on the mechanisms underlying traditional Chinese medicine.

<table><tbody><tr><td>Camera</td><td>Nikon</td><td></td><td></td></tr><tr><td>Capsaicine</td><td>Sigma</td><td>211275</td><td></td></tr><tr><td>CL-100 temperature controller</td><td>Warner Instruments</td><td></td><td></td></tr><tr><td>Cyclopiazonic Acid (CPA)</td><td>Sigma</td><td>C1530</td><td></td></tr><tr><td>DG-4 light</td><td>Sutter Instrument Company</td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)&amp;nbsp;</td><td>Amresco</td><td>231</td><td></td></tr><tr><td>DPBS</td><td>Thermofisher</td><td>14190144</td><td></td></tr><tr><td>Fluorescence imaging software (MetaFluor, Paid software)&amp;nbsp;</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Fura-2/AM</td><td>Invitrogen</td><td>F1201</td><td></td></tr><tr><td>HBSS buffer</td><td>Gibco</td><td>14175103</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Invitrogen</td><td>L3000008</td><td></td></tr><tr><td>Pluronic F-127</td><td>&amp;nbsp;Beyotime</td><td>ST501</td><td></td></tr><tr><td>poly-D-lysine</td><td>&amp;nbsp;Beyotime</td><td>ST508</td><td></td></tr><tr><td>SC-20 liquid circulation heating/cooling device&amp;nbsp;</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>White-light source</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

single-cell calcium imaging for studying the activation of calcium ion channels

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca2+ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca2+) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. By measuring the 340/380 ratio, the Ca2+ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca2+ changes in multiple cells. The results are stored in &#8220;.XLSX&#8221; format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca2+ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca2+ imaging.

Temperature response detection 
Primary keratinocyte 
Primary keratinocytes were isolated from newborn mice and prepared according to established protocols10. These cells were seeded into 24-well plates containing glass slides. Following the loading of the Fura-2 probe, the focus was adjusted under the microscope at a wavelength of 380 nm to achieve a clear visualization of cell morphology, as illustrated in Figure 2A. If the probe was not successfully loaded during the process, the cells would not be visible. The extracellular buffer in the chamber was continuously replaced through a perfusion system, ensuring optimal conditions for the cells. The temperature control system was connected to the perfusion system, enabling the detection of cellular thermal responses by controlling temperature changes in the extracellular buffer. This process involved gradually heating the extracellular buffer and then gradually cooling it.
The single-cell Ca2+ imaging system was utilized to monitor changes in intracellular Ca2+ concentration under various experimental conditions. As shown in Figure 2B, keratinocytes displayed a distinct thermal response when subjected to heating, as well as a noticeable response during the cooling phase. Notably, this temperature response disappeared when the STIM1 gene, which is primarily responsible for this response, was deleted (Figure 2C)10. Single-cell Ca2+ imaging allows for the observation of real-time changes in intracellular Ca2+ concentration in response to various external stimuli, enabling detailed analysis of cellular behavior and signaling.
HEK293T cells overexpressing the target plasmids 
HEK293T cells were seeded into a 24-well plate containing glass slides according to the experimental method. The cells were then overexpressed with STIM1/Orai1, and their mediated thermal response was detected using the same method as described above for primary cells. In the case of cells overexpressing the target plasmid, successful transfection was confirmed by selecting cells that expressed the corresponding plasmid, as indicated by red or green fluorescent proteins carried by the plasmid under TRITC or FITC wavelengths. For this experiment, STIM1-GFP/Orai1-DsRed plasmids were utilized, with GFP detected under FITC, emitting green fluorescence, and DsRed detected under TRITC, emitting red fluorescence. Cells overexpressing empty mCherry (TRITC) were used as negative controls. As shown in Figure 3A, fluorescent cells observed under FITC indicated successful expression of STIM1, enabling the selection and recording of their thermal response during the heating and cooling processes. Cells that were not transfected served as the negative control. Figure 3B demonstrates that STIM1/Orai1 exhibited a significant thermal response during the cooling process, contrasting with the negative control shown in Figure 3C.
Drug response detection 
Single-cell Ca2+ imaging technology can also be used to detect cell responses to compounds. The cell preparation for thermal response detection is the same as described above. The difference between drug response and thermal response lies in the fact that thermal response usually requires the activation of cells by changing the temperature of the extracellular buffer through a perfusion system, while compounds can be administered either through perfusion or by directly adding them using a pipette. Direct addition with a pipette can effectively conserve the amount of compound used; however, the resulting curve may not always be as visually appealing as that obtained through perfusion.
Taking Figure 4 as an example, Figure 4A shows the store-operated calcium entry (SOCE) response of STIM1/Orai1, where the extracellular Ca2+ concentration and Cyclopiazonic Acid (CPA) content are sequentially changed through a perfusion system to induce SOCE. In Figure 4B, the TRPV1 agonist capsaicin is directly added using a pipette to cells overexpressing the TRPV1 plasmid. Capsaicin can activate TRPV1 and induce extracellular Ca2+ influx.
<img alt="Fura-2 calcium imaging; fluorescence, ester hydrolysis, intensity at 340/380 nm, diagram." class="xfigimg" src="/files/ftp_upload/67412/67412fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1">
Figure 1: Schematic diagram of Fura-2 AM. Fura-2 is a commonly used Ca2+ fluorescence probe in cell biology that selectively binds to Ca2+ in a 1:1 ratio and emits fluorescence. Due to its highly polar nature as an acidic compound, Fura-2 cannot enter cells. To enhance cell permeability, it is conjugated with acetoxymethyl (AM) on its negative group to form Fura-2/AM. This modification increases its ester solubility and eliminates the negative charge. Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. Upon binding, the maximum excitation wavelength shifts from 380 nm to 340 nm (when saturated with Ca2+). The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. <a href="https://www.jove.com/files/ftp_upload/67412/67412fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescent microscopy, calcium imaging; Fura-2 ratio graphs showing cells' ATP response over time." class="xfigimg" src="/files/ftp_upload/67412/67412fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Temperature response detection of primary keratinocytes. (A) Fura-2 staining of keratinocytes from newborn mice, observed under a 20x objective lens. (B,C) Temperature response of WT keratinocytes (B) and STIM1 knockout keratinocytes (C) using a single-cell Ca2+ imaging system. Magnification: 20x. <a href="https://www.jove.com/files/ftp_upload/67412/67412fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="STIM1-GFP fluorescence microscopy, Fura-2 ratio graphs, calcium signaling analysis, heat and ATP." class="xfigimg" src="/files/ftp_upload/67412/67412fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Temperature response detection of STIM1-GFP/Orai1-Dsred overexpressed in HEK293T cells. (A) A representative figure showing positive cells expressing STIM1-GFP under FITC. (B,C) Temperature response of HEK293T cells expressing the respective plasmid detected by a single-cell Ca2+ imaging system. Magnification: 20x. <a href="https://www.jove.com/files/ftp_upload/67412/67412fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Calcium signaling graph, Fura-2 ratio changes, STIM1-GFP/Orai1-DsRed, TRPV1-mRuby, capsaicin effect." class="xfigimg" src="/files/ftp_upload/67412/67412fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Drug response detection using single-cell Ca2+ imaging. (A) SOCE response of HEK293T cells overexpressing STIM1/Orai1. The extracellular buffer is changed from Ca2+-containing to Ca2+-free using a perfusion system. In the absence of Ca2+, CPA is added to deplete Ca2+ from the endoplasmic reticulum. The extracellular buffer is then replaced with a Ca2+-containing buffer to induce extracellular Ca2+ influx, representing store-operated Ca2+ entry. Single-cell Ca2+ imaging is utilized to dynamically record the changes in cytosolic Ca2+ concentration induced by these processes. (B) HEK293T cells overexpressing TRPV1-mRuby, where mRuby is a fluorescent protein used to check the transfection condition of the TRPV1 protein. Capsaicin is added using a pipette while the Ca2+ influx is recorded by single-cell Ca2+ imaging. <a href="https://www.jove.com/files/ftp_upload/67412/67412fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...

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Research

JoVE Journal

Neuroscience

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Learn single-cell calcium imaging using Fura-2/AM dye to monitor TRPV1 channel activation and thermal responses in keratinocytes and HEK293T cells. This method enables real-time quantitative analysis of calcium signaling for pain and temperature sensation research.

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

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