Raz Zarivach is supported by the Israel Science Foundation (grant no. 163/22). Tomer Eli Ben Yosef and Arie Moran are supported by the Israel Science Foundation (grant no. 2047/20). We would like to thank Daniel Gitler and his group at Ben-Gurion University for their cooperation, support, and expertise.

1. Cell transfection
<ol>
	<li>Culture HEK293T cells in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1x penicillin/streptomycin (see Table of Materials) in a humidified incubator at 37 &#176;C/5% CO2 until confluence on a 10 cm plate (8.8 x 106 total cells).</li>
	<li>Place one 13 mm coverslip in each of the wells of a 12-well plate. Dilute 0.44 x 106 trypsinized cells from step 1.1 in 12 mL of complete DMEM. Mix well by pipetting up and down three to five times. Fill each well with 1 mL of the mixed solution. Grow in a humidified incubator at 37 &#176;C/5% CO2 overnight.</li>
	<li>Replace the complete DMEM in each well with 1 mL of serum-free DMEM (see Table of Materials) in each well. Return the 12-well plate to the humidified incubator as in step 1.1.</li>
	<li>For each transfection well, dilute 1 &#181;g of pAAV2 plasmid containing the protein of interest tagged with mCherry fluorescent protein (Supplementary File 1) in 100 &#181;L of serum-free DMEM in a 1.5 mL tube.</li>
	<li>Add 3 &#181;g of polyethyleneimine (PEI, see Table of Materials) to 100 &#181;L of serum-free DMEM per transfection, with each transfection in a different 1.5 mL tube. The plasmid:PEI ratio should be 1:3 (&#181;g:&#181;g).</li>
	<li>Vortex both tubes from step 1.4&#160;and step 1.5&#160;for 10 s, and leave to rest at room temperature for at least 5 min.</li>
	<li>Mix one part (100 &#181;L per well) of the DNA solution from step 1.4 with one part (100 &#181;L per well) of the PEI solution from step 1.5. Vortex the final solution for 10 s, and leave it to rest at room temperature for at least 20 min and for up to 6 h.</li>
	<li>Take the 12-well plate from the incubator. Vortex the final solution from step 1.7, and add 200 &#181;L to each of the wells in the 12-well plate from step 1.3. Return the 12-well plate to the humidified incubator (step 1.1).</li>
	<li>After 3 h, replace the medium in each well with 1 mL of complete DMEM. Return the 12-well plate to the humidified incubator as in step 1.1.</li>
	<li>After 2 days, take the transfected cell culture from the 37 &#176;C/5% CO2 incubator, and place it on an inverted fluorescence microscope using 10x magnification.</li>
	<li>Using the microscope focus wheel, focus on the cells while using brightfield light.</li>
	<li>Switch to the fluorescent mCherry excitation (587 nm) and emission (610 nm) wavelengths, and turn off the brightfield light. Check for the fluorescence of the cells to confirm the expression of ZnT1 mCherry.</li>
	<li>Prepare the dye loading solution.
	<ol>
		<li>Take an aliquot of 4 &#181;L of zinc-specific fluorescent dye (see Table of Materials) in its acetoxymethyl (AM) ester form, dissolved in DMSO (1 &#181;g/&#181;L), from a light-protected stock stored in a &#8722;20&#160;&#176;C freezer. This form allows the dye to enter the cell through the plasma membrane by simple diffusion.</li>
		<li>Add 4 &#181;L of 10% pluronic acid (or 2 &#181;L of 20% pluronic acid, see Table of Materials) to the aliquot from step 1.13.1. Mix the solution well by pipetting up and down three times, and then add all 8 &#181;L to a 1.5 mL tube.</li>
		<li>Add 750 &#181;L of Ringer&#39;s solution (prepared in-house, see Table of Materials for composition) supplemented with 1 mg/mL (~0.1%) bovine serum albumin to the tube from step 1.13.2, and vortex vigorously. Add another 750 &#181;L of the same solution, and vortex again to ensure maximum mixing.</li>
	</ol>
	</li>
	<li>Add 750 &#181;L of the final solution from step 1.13.3&#160;to two wells in a new 6-well culture plate. Cover with aluminum foil. 
	NOTE: To avoid bleaching, the 6-well plate is always covered with aluminum foil from this step.</li>
	<li>Using a fine tweezer, take up to four replicate coverslips from the transfected cell culture plate, and place them in the first filled well (up to four slides per well) of the new 6-well plate from step 1.14. Repeat this process for the second filled well. 
	NOTE: All the coverslips in the same filled well must be from the same condition. However, each well can contain different conditions.</li>
	<li>Cover with aluminum foil, and gently shake for 15-20 min.</li>
	<li>Remove the dye loading solution, and replace it with a new washing solution of Ringer&#39;s plus albumin, as used in&#160;step 1.13.3. Cover with aluminum foil. Leave to shake again for 20 min. This allows the cleavage of the AM ester by the intracellular esterases.</li>
</ol>
2. Microscope preparation
<ol>
	<li>Arrange the necessary tools (see Table of Materials) as mentioned: an inverted fluorescence microscope capable of detecting GFP and mCherry fluorophores, a perfusion system that allows switching between at least two solutions, a suction system, and a perfusion chamber.</li>
	<li>Turn on the microscope, its light source, and the camera. Turn on the suction system.</li>
	<li>Wash the perfusion system. Wash the first chamber with Ringer&#39;s solution and the second chamber with Ringer&#39;s solution containing 7 &#181;M zinc solution supplemented with 7 &#181;M pyrithione (zinc ionophore, see Table of Materials). To avoid air bubbles or gaps, leave some liquid in each chamber. 
	NOTE: Since both containers are connected to the same tube hooked to the perfusion chamber, always ensure that the shared segment is washed with Ringer&#39;s solution.</li>
	<li>Close the taps, and fill the appropriate chambers with Ringer&#39;s solution and Ringer&#39;s zinc solution, as in step 2.3.</li>
</ol>
3. Sample preparation
<ol>
	<li>Take the perfusion chamber, and place it with the narrow side of the groove facing upward. 
	NOTE: The groove is a hole in the middle of the perfusion chamber. It allows the perfusion fluid access to the cells. One side of the groove is narrow, and the opposite side is wide.</li>
	<li>Apply a sealing silicone (see Table of Materials) around the groove. Clean any sealing silicone from the groove using a pipette tip. 
	NOTE: Ensure there is enough silicone seal around the groove to seal a 22 mm coverslip.</li>
	<li>Using a fine tweezer, take a coverslip from the washing solution, and place it on top of the groove with the cells facing down. This way, the cells will be exposed to the solution perfusing the groove during the experiment.</li>
	<li>Place a 22 mm coverslip on top of the 13 mm coverslip, and tighten it using the tweezer. Take care not to crack the coverslips.</li>
	<li>Flip the chamber, and press on it to release all the present liquids. Fill the groove with 100 &#181;L of Ringer&#39;s washing solution, and press again to ensure no leakage. 
	NOTE: If a leakage is detected, apply a sealing agent at the site of the leakage, and retest.</li>
	<li>Mount the perfusion chamber onto the platform, and secure it. Place the perfusion and suction tubes to allow perfusion over the cells in the groove.</li>
	<li>Change the perfusion rate to approximately 2 mL/min, and turn on the perfusion of the Ringer&#39;s solution. Ensure the perfusion system is working with no leakage or spillover.</li>
</ol>
4. Measurement preparation
<ol>
	<li>Open the imaging software (see Table of Materials) by double-clicking on the icon. Log in with the relevant credentials. Choose the attached camera, and press Ok.</li>
	<li>Set the microscope magnification to 10x using the button on the left side of the microscope.</li>
	<li>Choosing live view and using the joystick, move the platform to focus on the cells in the groove.</li>
	<li>While the perfusion is on, turn off the lights and change the wavelength to mCherry by pressing the dedicated button in the interface. Adjust the focus using the microscope focus wheel.</li>
	<li>Once the microscope is focused on the cells, move the platform using the joystick to allow the selection of the appropriate cell patches. 
	NOTE: A suitable area is considered an area with at least 10 cells for an ROI and an empty area for background.</li>
	<li>Select the Turn&#160;ROIs On/Off drop-down menu, and choose Draw Circular ROIs.</li>
	<li>Draw ROIs of cell clusters with the mCherry-expressing cells (indicates the expression of ZnT1).</li>
	<li>From the same toolbar as step 4.6, click the Turn background ROIs On/Off button, and adjust the location and size of the background ROI to an area with no cells at all.
	<ol>
		<li>Check with the mCherry and EGFP wavelengths to ensure no cells are in the selected background ROI. 
		NOTE: The wavelengths were adjusted so that mCherry used an excitation wavelength of 520 nm and an emission wavelength of 610 nm and EGFP used an excitation wavelength of 470 nm and an emission wavelength of 520 nm.</li>
	</ol>
	</li>
	<li>Select the Wavelength (&#955;) sub-tab. Mark its checkbox, and ensure that the GFP wavelength is present and its checkbox marked. If not, add and mark it.</li>
	<li>Select the Duration sub-tab. Define the measurement interval as every 5 s, and change the number of intervals to suit the experiment duration.</li>
	<li>In the Focus section on the microscope panel below the eyepiece, click on the On button to enable the perfect focus system (PFS).</li>
	<li>Using the PFS focus wheel, adjust the focus. In the software interface, Under ND Acquisition, ensure PFS on is ticked.</li>
	<li>Click on Run now.</li>
</ol>
5. Experimental procedure
<ol>
	<li>Start with a 90 s baseline period measurement using Ringer&#39;s solution perfusion.</li>
	<li>After the baseline period ends, turn off the Ringer&#39;s solution perfusion, and then turn on the Ringer&#39;s zinc solution perfusion. Mark the switching of the solution by clicking the red flag on the bottom right of the main interface panel. A rise in fluorescence is expected to appear.</li>
	<li>Once the fluorescence rise starts to saturate, change back to perfusion with Ringer&#39;s solution. Turn off the Ringer&#39;s zinc solution perfusion, and then turn on the Ringer&#39;s solution perfusion.</li>
	<li>Wait until the experiment time expires. A noticeable yet steady decrease in fluorescence is expected if the ZnT1 is working properly.</li>
</ol>
6. Data export
<ol>
	<li>To export the data with baseline subtraction, press the Subtract Baseline button, and view the changes in the &#34;ND acquisition window&#34;.</li>
	<li>Click on the Export button in the software interface in the &#34;ND acquisition window&#34;. An excel datasheet will open. Save to the desired location.</li>
</ol>
7. Data analysis
<ol>
	<li>For each ROI, create an average baseline fluorescence from the first 90-100 s.</li>
	<li>Express the fluorescence calculated for each ROI as a percentage of the background for that ROI.</li>
	<li>Create a row average of all the ROIs, and plot the result as a line graph. This creates an average of all the ROIs as a function of time.</li>
	<li>Using the linear fit function, select the initial rate for the decrease in fluorescence following the wash with Ringer&#39;s solution. The slope value of the equation correlates with the transport rate.</li>
</ol>

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay

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The authors declare no conflicts of interest.

The above-described method allows for the direct measurement of the intracellular zinc concentration with high temporal resolution. Compared to other methods, this method involving monitoring changes in intracellular Zn2+ can substantially decrease background noise. In addition, the dye&#39;s selectivity for zinc eliminates potential cross-interactions with other metal cations18,19. Finally, its lack of immediate cytotoxicity enables the testing of liv...

Zinc is an essential trace element in the cellular milieu. It incorporates one-third of all proteins and is involved in various cellular processes, such as catalysis1, transcription2, and structural motifs3. However, despite being redox-inert, high zinc concentrations are toxic to the cell, which is why no mammalian organism has survived without the presence of mechanisms regulating zinc homeostasis. In mammals, three mechanisms are responsible for this process: (1) metallothioneins, which are cytosolic cysteine-rich proteins that bind zinc at a high affinity, thus preventing excess free cytosolic zinc4; (2) Zrt/Irt-like proteins (ZIPs), which are zinc transporters responsible for zinc influx into the cytosol through the plasma membrane or from intracellular organelles4,5,6,7,8; and (3) ZnTs, which are a mammalian subset of the ubiquitous cation diffusion facilitator (CDF) family and are zinc transporters, as they extrude zinc from the cytosol across the plasma membrane or into the intracellular organelles4,5,6,7,8,9. Due to the importance of zinc to cellular metabolism, it is vital to understand cellular zinc dynamics.
Previous methods to assess zinc dynamics depended on assessing the expression levels of mRNA under different zinc conditions by correlating them with cellular zinc measurements of fixed tissues or cells10,11,12. These methods include chemical detection and immunohistochemistry staining. However, these methods yield only indirect measures and, thus, determine only an offline correlation between intracellular zinc concentration and the expression of zinc transporters. Consequently, these methods cannot infer any parameters requiring high temporal resolution.
A more direct measurement of Zn2+ transport uses radioactive isotopes of zinc13. This method relies on the measurement of radiolabeled Zn2+ to monitor zinc transport and its kinetics. However, due to the importance of zinc to cellular homeostasis, multiple cellular processes regulate intracellular zinc concentration. Among these are extracellular binding and several transport systems that work in concert to maintain tight control of intracellular Zn2+ levels. The combination of these processes creates considerable background noise, which makes it difficult to test individual zinc-related transport functions.
This article demonstrates a method to directly monitor the zinc transport rate by measuring the intracellular free zinc concentration using a zinc-specific fluorescent dye, FluoZin-3. The dye has high specificity for Zn2+ and little interference from other divalent cations, such as calcium. In addition, in its ester form, it enters the cells by nonionic diffusion and is then trapped due to the activity of intracellular di-esterase. Thus, its fluorescence is correlated primarily with the free cytosolic zinc concentration. These experiments were conducted to study the structure-function relationship of zinc transporter 1 (ZnT1), a member of the ZnT family.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm plate</td>
			<td>greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate</td>
			<td>greiner bio-one</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>13 mm coverslips</td>
			<td>Superior Marienfeld</td>
			<td>111530</td>
			<td></td>
		</tr>
		<tr>
			<td>22 mm cover slides</td>
			<td>Superior Marienfeld</td>
			<td>101050</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>greiner bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>bioWorld</td>
			<td>22070008</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride anhydrous, granular</td>
			<td>Sigma Aldrich</td>
			<td>C1016</td>
			<td>Concentration in Ringer solution: 1 mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Glentham Life Science</td>
			<td>GC6947</td>
			<td>Concentration in Ringer solution: 10 mM</td>
		</tr>
		<tr>
			<td>Dubelco&#8217;s Modified Eagle Media (DMEM)&#160;</td>
			<td>Sartorius</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti inverted microscope</td>
			<td>Nikon</td>
			<td>TI-DH</td>
			<td>Discontinued. Replaced by Eclipse Ti2</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cytiva</td>
			<td>SH30088.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine tweezers</td>
			<td>Dumont</td>
			<td>0203-55-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluozin-3AM</td>
			<td>Invitrogen</td>
			<td>F24195</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Penicillin-Streptomycin 100x solution</td>
			<td>Cytiva</td>
			<td>SV30010&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LED illumination system</td>
			<td>CoolLED</td>
			<td>pE-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Biological Industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Merck</td>
			<td>1.05833</td>
			<td>Concentration in Ringer solution: 0.8 mM</td>
		</tr>
		<tr>
			<td>N[2-Hydroxyethyl]piperazine-N&#39;-[2-ethanesulfonic acid] (HEPES)</td>
			<td>Formedium</td>
			<td>HEPES10</td>
			<td>Concentration in Ringer solution: 10 mM</td>
		</tr>
		<tr>
			<td>Neo 5.5 sCMOS camera</td>
			<td>ANDOR</td>
			<td>DC-152Q-FI</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements imaging software</td>
			<td>Nikon</td>
			<td>AR</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic acid F-127</td>
			<td>Millipore</td>
			<td>540025</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium chloride</td>
			<td>Bio-Lab</td>
			<td>163823</td>
			<td>Concentration in Ringer solution: 5.4 mM</td>
		</tr>
		<tr>
			<td>Pyrithione</td>
			<td>Sigma Aldrich</td>
			<td>H3261</td>
			<td>Concentration in Ringer zinc solution: 7 &#956;M</td>
		</tr>
		<tr>
			<td>Silicone Grease Kit</td>
			<td>Warner Instruments</td>
			<td>W4 64-0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Bio-Lab</td>
			<td>190305</td>
			<td>Concentration in Ringer solution: 120 mM</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td></td>
			<td></td>
			<td>Concentration in Ringer zinc solution: 7 &#956;M</td>
		</tr>
		<tr>
			<td></td>
			<td>Sigma Aldrich</td>
			<td>31665</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing mammalian zinc transporters using an in vitro zinc transport assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

My research focuses on the structure-function relationship of mammalian zinc transporters, with special emphasis on the regulation mechanism. Due to the importance of zinc ion to cellular function, it is challenging to obtain accurate measurement of cellular zinc dynamics. Our protocol addresses the low signal-to-noise ratio and low temporal resolution of zinc dynamics.This method allows testing of zinc influx across the cell membrane, with high temporal resolution and considerably less background noise and cross-interactions by other divalent cations. We found that the unstructured extension of the mammalian transporter ZnT1 one C-terminal domain is not involved in the zinc transport function. This allows researchers to focus on other domains of the ZnT1 protein for zinc transport regulation.

ZnT1 is a mammalian zinc transporter located on the cell plasma membrane13. It is a member of the cation diffusion facilitator (CDF) protein family that extrudes zinc from the cytosol to the extracellular millieu14. ZnT1 has a two-domain architecture: the transmembrane domain, which transports the ions across the membrane, and a C-terminal domain14. Unlike other known CDF proteins, ZnT1 has an extended unstructured C-terminal domain (USCTD). The role...

Watch this Scientific Journal Video about Exploring Cellular Zinc Regulation Through ZnT1 Functionality at JoVE.com

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...

characterizing-mammalian-zinc-transporters-using-an-vitro-zinc

Research

JoVE Journal

Biology

792 Views.  Ben-Gurion University. My research focuses on the structure-function relationship of mammalian zinc transporters, with special emphasis on the regulation mechanism. Due to the importance of zinc ion to cellular function, it is challenging to obtain accurate measurement of cellular zinc dynamics. Our protocol addresses the low signal-to-noise ratio and low temporal resolution of zinc dynamics.This method allows testing of zinc influx across the cell membrane, with high temporal resolution and considerably les...

Video: Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

Zinc-Specific Staining of Transfected HEK293T Cells

Begin by analyzing transfected HEK293T cells using an inverted fluorescence microscope. Once the cells are focused using bright-field light, switch to fluorescent mCherry excitation wavelength of 587 nanometers and emission wavelengths of 610 nanometers. After turning off the bright-field light, check for the fluorescence of the cells to confirm the expression of ZnT1 mCherry.To prepare dye-loading solution, take four microliters of zinc-specific fluorescent dye solution. Then add four microliters of 10%pluronic acid. Mix the solution by pipetting up and down, and transfer it to a 1.5-milliliter tube.Add 750 microliters of washing solution into the prepared solution and vortex the tube vigorously. Again, add 750 microliters of wash solution. Once vortexed, add 750 microliters of this solution into two wells of a new six-well culture plate and cover it with aluminum foil.Using tweezers, transfer a 13-millimeter cover slip from the previously cultured HEK293T cell plate into a six-well plate. After covering the plate with foil, gently shake it for 15 to 20 minutes and replace the dye-loading solution with the prepared washing solution. Once the plate is covered, shake it again for 20 minutes.

Sample Preparation and Fluorescence Measurement of Stained Transfected HEK293T Cells

To begin, turn on the microscope, light source, camera, and suction system. For washing the perfusion system, use Ringer's solution in the first chamber and Ringer's zinc solution supplemented with pyrithione in the second chamber. After closing the tap, fill the chambers with the same solutions.To begin sample preparation, place the perfusion chamber with the narrow side of the groove facing up and apply a sealing silicone around the groove. Then, using a fine tweezer, transfer a 13-millimeter coverslip from the washing solution on top of the groove with cells facing down. Place a 22-millimeter coverslip on top of the 13-millimeter coverslip and tighten it using the tweezer, taking care not to crack the coverslips.Flip the chamber and press it to release the liquids. Then, fill the groove with 100 microliters of Ringer's washing solution and press again, ensuring no leakage. Mount and secure the perfusion chamber onto the platform.Then, attach the perfusion and suction tubes for perfusion over the cells in the groove. Change the perfusion rate to approximately two milliliters per minute and turn on Ringer's solution perfusion. For measurements, open the imaging software.Set the microscope magnification to 10X using the left side button of the microscope. After choosing Live View, use the joystick to move the platform and focus on the cells in the groove. Select the appropriate wavelength for mCherry.Once the cells are focused, move the platform for the selection of appropriate cell patches. Select the Draw ROIs dropdown menu. Choose Draw Circular ROIs and draw ROIs on cell clusters with the mCherry-expressing cells.Create new ROIs beyond the first by holding down the Shift button and then by pressing and dragging the existing ROIs. Select Draw Square Background ROI from the dropdown menu and adjust the location and size of the background ROI where cells are not present. After ensuring the absence of cells in the background ROI, go to the ND Acquisition tab.Select the Wavelength subtab and mark its checkbox. Ensure that the checkbox for GFP wavelength is also marked. Click on the Time subtab and define the measurement interval as every five seconds and duration as 30 minutes.On the microscope panel, press the On to enable the Perfect Focus System, or PFS. Under ND Acquisition, ensure PFS on is ticked. Adjust the focus and click Run now.Start baseline period measurement for 90 seconds using Ringer's solution perfusion. After 90 seconds, switch from the Ringer's solution perfusion to the Ringer's zinc solution perfusion, and click the red flag on the bottom right of the main interface panel once the fluorescence rise starts to saturate. Change back to perfusion with Ringer's solution until the experiment time expires.To export the data with baseline subtraction, press the Background Correction button and view the changes in the ND Acquisition window. Click the Export button to save the data in the desired location. An increase in fluorescence resulted from increased intracellular zinc concentration, while a decrease indicated the activity of ZnT-1 transporting zinc across the cell membrane.The wild type ZnT-1 had a smoother fluorescence curve than the mutant, relating to the variability in the cellular responses to zinc load. A box plot comparing the activities of wild-type and delta USCTD showed similar average transport rates, indicating a lack of difference between the wild type and the mutant. However, the difference in the spread may indicate a functional difference.