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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-responsive+transcription+factors+that+regulate+iron,+zinc,+and+copper+homeostasis+in+eukaryotic+cells.">Metal-responsive transcription factors that regulate iron, zinc, and copper homeostasis in eukaryotic cells.</a> Eukaryotic Cell. 3 (1), 1-13 (2004).</li><li>Maret, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+Biochemistry:+From+a+single+zinc+enzyme+to+a+key+element+of+life.">Zinc Biochemistry: From a single zinc enzyme to a key element of life.</a> Advances in Nutrition. 4 (1), 82-91 (2013).</li><li>Kimura, T., Kambe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+metallothionein+and+ZIP+and+ZnT+transporters:+An+overview+and+perspective.">The functions of metallothionein and ZIP and ZnT transporters: An overview and perspective.</a> International Journal of Molecular Sciences. 17 (3), 336 (2016).</li><li>Kambe, T., Hashimoto, A., Fujimoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+ZIP+and+ZnT+zinc+transporters+in+human+health+and+diseases.">Current understanding of ZIP and ZnT zinc transporters in human health and diseases.</a> Cellular and Molecular Life Sciences. 71 (17), 3281-3295 (2014).</li><li>Kambe, T., Tsuji, T., Hashimoto, A., Itsumura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological,+biochemical,+and+molecular+roles+of+zinc+transporters+in+zinc+homeostasis+and+metabolism.">The physiological, biochemical, and molecular roles of zinc transporters in zinc homeostasis and metabolism.</a> Physiological Reviews. 95 (3), 749-784 (2015).</li><li>Hara, T., Yoshigai, E., Ohashi, T., Fukada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+transporters+as+potential+therapeutic+targets:+An+updated+review.">Zinc transporters as potential therapeutic targets: An updated review.</a> Journal of Pharmacological Sciences. 148 (2), 221-228 (2022).</li><li>Kambe, T., Taylor, K. M., Fu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+transporters+and+their+functional+integration+in+mammalian+cells.">Zinc transporters and their functional integration in mammalian cells.</a> Journal of Biological Chemistry. 296, 100320 (2021).</li><li>Huang, L., Tepaamorndech, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SLC30+family+of+zinc+transporters+-+A+review+of+current+understanding+of+their+biological+and+pathophysiological+roles.">The SLC30 family of zinc transporters - A review of current understanding of their biological and pathophysiological roles.</a> Molecular Aspects of Medicine. 34 (2), 548-560 (2013).</li><li>Lovell, M. A., Smith, J. L., Xiong, S., Markesbery, W. 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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 live cellular processes19.
However, this method has certain limitations. First, detecting intracellular dynamics can be relatively more challenging with this protocol, since the dye used in this method is designed to be trapped in the cell but cannot be directed to regions or specific processes. Other fluorescent dyes can be directed to a specific area in the cells but, unfortunately, have a much lower dynamic range20. Second, the exposure of the dye to light causes a decrease in fluorescence due to bleaching, which restricts the time of exposure to light and, consequently, the active window for running the experiment. Third, the dye loading in this protocol is cumbersome and time-consuming when compared to using other dyes, especially genetically encoded dyes21. Finally, even after cleaving the esters, the dye leaks, leading to a Zn2+-independent reduction in fluorescence22.
Several steps should be taken to ensure the experiment&#39;s success. First, since transfection can be unequally distributed, the selected cells should contain the protein of interest. To avoid dye bleaching, it is important to keep the loaded cells protected from light. When running the experiment, the perfusion rate should be sufficient to complete the experiment without causing the detachment of the cells from the coverslip. In the case of multiple runs, keeping the same reservoirs for the Ringer&#39;s solution and Ringer&#39;s zinc solution is recommended. The dye loading time and wash time can be adjusted according to initial experiments. Zinc loading into the cells via perfusion with pyrithione can take from 1 min to 5 min, as assessed by monitoring the Zn2+-induced fluorescence with time.
Despite these difficulties, this method allows us, for the first time, to study the dynamics of the Zn2+ extruding process. It simplifies the process and creates a direct way to better establish causal links compared to the previous methods23,24.

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.

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 of the USCTD is currently unknown. We used the protocol above to compare ZnT1 WT to ZnT1 without the USCTD to assess its involvement in zinc transport activity.
HEK 293T cells were transfected with the pAAV2 plasmid containing either ZnT1 WT or ZnT1 without the unstructured C-terminal (&#916;USCTD) and tested the transport activity rate as described above. Supplementary Figure 1 shows that both versions of ZnT1 express and localize to the plasma membrane without any issues. Figure 1 depicts the typical results for an example of such an experiment. An increase in fluorescence is the result of an increase in the intracellular Zn2+ concentration, while a decrease is a result of the activity of ZnT1 transporting Zn2+ across the cell membrane (individual graphs, including standard errors, are found in Supplementary Figure 2 and Supplementary Figure 3). It is evident from this figure that ZnT1 WT has a smoother fluorescence curve than the mutant. However, this is not an inherent differentiating feature but is related to variability in the cellular responses to zinc load, which has been observed in previous experiments. Figure 2 shows a box plot summarizing the results from 15-17 such experiments. From the figure, it is clear that &#916;USCTD presents a wider spread of transport rates compared to WT. While this can be attributed to the cellular response variability previously mentioned, it could also indicate a functional difference. For example, it may imply that the ZnT1 USCTD segment is a modulator of the transport rate. Clearly, there is no visible difference between the Zn2+ extruding activities of WT and &#916;USCTD. Based on the statistical analysis, the data were not normally distributed (WT dataset, Shapiro-Wilk test15, statistic = 0.72404, p value = 0.0004), so two sample non-parametric tests were used for comparisons between the datasets. The results showed that there was no statistical difference between the transport rates (Mann-Whitney16 U test: U = 132, Z = 0.15105, Asymp. Prob&#62;|U| = 0.87994; Kolmogorov-Smirnov17 test: D = 0.27059, Z = 0.76384, Exact Prob&#62;|D|= 0.5161).
Due to the substantial energetic cost of creating the unstructured extension (~80 amino acids), it is reasonable to assume that this domain serves a cellular function. However, a cellular function linked to this domain has not been identified so far. This domain is unique to ZnT1 and is not found in any other members of the ZnT family. As such, it may be due to the fact that, unlike other ZnTs, ZnT1 is located at the plasma membrane, while except for ZnT10, all other members are expressed in internal organelles.
The raw data and subsequent graphs shown here are based on 5 s interval measurements of the fluorescence caused by the zinc-sensitive dye. This means that in a relatively short time frame (minutes), a visualization of time-dependent zinc transport can be generated with high temporal resolution. The analysis should include a baseline period normalization since the dye is loaded into the cells prior to zinc loading and may have initial intracellular fluorescence caused by a small amount of free zinc. In addition, the zinc dye only becomes fluorescently active after cleavage by the intracellular esterase. Therefore, it is recommended to allow some time for the de-esterification process to occur for most of the dye in order to avoid irregular fluorescence patterns caused by dye ester cleavage throughout the experiment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65217/65217fig01.jpg" /> 
Figure 1: Zn2+ extruding activities of WT and &#916;USCTD using the zinc transport assay. The x-axis is the time in seconds. The y-axis is the fluorescence change expressed as a percentage of baseline. The line graphs are the fluorescence change as a function of time. The data are averaged over &#62;10 ROIs. Individual graphs with standard error are shown in Supplementary Figure 2 and Supplementary Figure 3. <a href="https://www.jove.com/files/ftp_upload/65217/65217fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65217/65217fig02.jpg" /> 
Figure 2: Comparison of the activities of ZnT1 WT and &#916;USCTD depicted as a box plot. Each data point represents a single experiment. The black line indicates the median score. The hollow square box indicates the mean score. The y-axis indicates the change in baseline percentage per second. <a href="https://www.jove.com/files/ftp_upload/65217/65217fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: HEK 293T cells transfected with (A) ZnT1 WT or (B) ZnT1 &#916;USCTD stimulated in the mCherry excitation (520 nm) and emission (610 nm) wavelengths. In both instances, there is expression and membrane localization of the ZnT1 variant. The microscope magnification is 20x. The exposure time is 100 ms. <a href="https://www.jove.com/files/ftp_upload/65217/Figure S1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: ZnT1 WT Zn2+ transporting activity with the standard error visualized. The x-axis is the time in seconds. The y-axis is the percentage fluorescence change from the baseline fluorescence. The black line is the mean fluorescence change. <a href="https://www.jove.com/files/ftp_upload/65217/Figure S2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: ZnT1 &#916;USCTD Zn2+ transporting activity with the standard error visualized. The x-axis is the time in seconds. The y-axis is the percentage fluorescence change from the baseline fluorescence. The red line is the mean fluorescence change. <a href="https://www.jove.com/files/ftp_upload/65217/Figure S3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 1: pAAV2 plasmid sequence. <a href="https://www.jove.com/files/ftp_upload/65217/DG84 pAAV2 hSyn ZnT1 mCherry.zip" target="_blank">Please click here to download this File.</a>

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

Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

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.

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 ...

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1.1K Views.  Ben-Gurion University. 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.

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

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

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An In vitro FluoroBlok Tumor Invasion Assay

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DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

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DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

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A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

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A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

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Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

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Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

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Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

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Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

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Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

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Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies.&#160;

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

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Titration of Human Coronaviruses Using an Immunoperoxidase Assay

An In vitro FluoroBlok Tumor Invasion Assay

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys₂-His₂ Zinc-finger Domains

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon