Name: Zinc-Specific Staining of Transfected HEK293T Cells
Uploaded: 2023-06-02T13:20:00
Description: Watch this Scientific Journal Video about Zinc-Specific Staining of Transfected HEK293T Cells at JoVE.com

zinc-specific staining of 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 Zinc-Specific Staining of Transfected HEK293T Cells at JoVE.com

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.

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 ...