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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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+airway+mucus+secretion+and+pathophysiology+of+hypersecretion.">Physiology of airway mucus secretion and pathophysiology of hypersecretion.</a> Respir Care. 52 (9), 1134-1146 (2007).</li><li>Mitra, R., Hasan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+Ca(2+)+entry+regulates+neuronal+gene+expression+and+function.">Store-operated Ca(2+) entry regulates neuronal gene expression and function.</a> Curr Opin Neurobiol. 73, 2022 (2022).</li><li>Landstrom, A. P., Dobrev, D., Wehrens, X. H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+and+cardiac+arrhythmias.">Calcium signaling and cardiac arrhythmias.</a> Circ Res. 120 (12), 1969-1993 (2017).</li><li>Zheng, C., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+deficiency+of+coagulation+factors+v+and+viii:+An+update.">Combined deficiency of coagulation factors v and viii: An update.</a> Semin Thromb Hemost. 39 (6), 613-620 (2013).</li><li>Ahmadian Elmi, M., Motamed, N., Picard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analyses+of+the+g+protein-coupled+estrogen+receptor+gper1+reveal+constitutive+links+to+endoplasmic+reticulum,+glycosylation,+trafficking,+and+calcium+signaling.">Proteomic analyses of the g protein-coupled estrogen receptor gper1 reveal constitutive links to endoplasmic reticulum, glycosylation, trafficking, and calcium signaling.</a> Cells. 12 (21), 2571 (2023).</li><li>Prakriya, 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=Orai1+is+an+essential+pore+subunit+of+the+crac+channel.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+capsaicin+receptor:+A+heat-activated+ion+channel+in+the+pain+pathway.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stim1+thermosensitivity+defines+the+optimal+preference+temperature+for+warm+sensation+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+thermoreceptor+trpm8+by+cooling+compounds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezo+proteins+are+pore-forming+subunits+of+mechanically+activated+channels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+analysis+of+stim1+thermal+sensation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATF6+safeguards+organelle+homeostasis+and+cellular+aging+in+human+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanosensitive+piezo1+channel+mediates+heart+mechano-chemo+transduction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Orai1-mediated+store-operated+Ca(2+)+channel/calpain+signaling+contributes+to+high+glucose-induced+podocyte+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+er+ion+homeostasis+maintained+by+an+er+anion+channel+clcc1+contributes+to+als-like+pathologies.">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><li>Gouin, O., 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=Trpv1+and+trpa1+in+cutaneous+neurogenic+and+chronic+inflammation:+Pro-inflammatory+response+induced+by+their+activation+and+their+sensitization.">Trpv1 and trpa1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization.</a> Protein Cell. 8 (9), 644-661 (2017).</li><li>Barreto-Chang, O. L., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+cortical+neurons+using+fura-2+am.">Calcium imaging of cortical neurons using fura-2 am.</a> J Vis Exp. (23), e1067 (2009).</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=A+lever-like+transduction+pathway+for+long-distance+chemical-+and+mechano-gating+of+the+mechanosensitive+piezo1+channel.">A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive piezo1 channel.</a> Nat Commun. 9 (1), 1300 (2018).</li></ol>

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 play...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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)&#160;</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)&#160;</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&#160;</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>&#160;Beyotime</td>
			<td>ST501</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>&#160;Beyotime</td>
			<td>ST508</td>
			<td></td>
		</tr>
		<tr>
			<td>SC-20 liquid circulation heating/cooling device&#160;</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 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 ...

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

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 ...

single-cell-calcium-imaging-for-studying-activation-calcium-ion

Research

JoVE Journal

Neuroscience

459 Views.  Beijing University of Chinese Medicine. 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.

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

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

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 ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...