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

405 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 ...

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 ...

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 ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Real-Time Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...