Name: Sample Preparation and Fluorescence Measurement of Stained Transfected HEK293T Cells
Uploaded: 2023-06-02T13:20:00
Description: Watch this Scientific Journal Video about Sample Preparation and Fluorescence Measurement of Stained Transfected HEK293T Cells at JoVE.com

sample preparation and fluorescence measurement of stained transfected hek293t cells

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

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.

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.

Watch this Scientific Journal Video about Sample Preparation and Fluorescence Measurement of Stained Transfected HEK293T Cells at JoVE.com

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.

sample-preparation-fluorescence-measurement-stained-transfected

Research

JoVE Journal

Biology

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.

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 ...

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.