Intracellular Zinc

Zinc, present throughout the body in its cationic form (Zn2+), is an essential micronutrient and an ubiquitous trace element found. Zinc is tightly regulated in the brain and has widespread function within neurons and in neurotransmission. Zinc is also a vital cofactor for the structural maintenance and stabilization of numerous proteins. Additionally, zinc plays a pivotal role amongst many cellular processes including enzymatic activity and gene regulation. It is no surprise, then, that the alteration of zinc homeostasis may cause the dysfunction of many organs and systems. For example, in the immune system abnormal zinc levels affect the differentiation, proliferation and function of inflammatory cells, including eosinophils, T and B cells, by altering signaling pathways.

It is also well established that zinc overexposure is toxic to neurons both in vitro and in vivo. The relationship between reactive oxidative species (ROS) and intracellular zinc has also proven to be intimately related. ROS production can lead to the release of zinc into cytoplasmic compartments, where it may not only impair mitochondrial function but also activate ROS-generating signaling cascades in the cytoplasm. In addition, adequate zinc levels are essential for correct immune responses and lower than normal zinc levels have been reported in many allergic inflammatory diseases, including atopic dermatitis, bronchial asthma, and chronic rhinosinusitis.

Intracellular Zinc Measurement Kits

---

Colorimetric

Currently, there are several commercially available kits used to measure intracellular zinc. In colorimetric assays, zinc binds with a ligand which can be measured using a microplate reader. Colorimetric assays can be used with many types of biological samples, including serum, plasma, cerebrospinal fluid, or urine, and have a strong detection sensitivity as low as 1 µM or lower.

In experimentation, first samples are prepared accordingly. Depending on starting material, they can be used directly or may require neutralization and/or deproteinization. Next, a standard curve is prepared via serial dilutions of the provided zinc standard. The samples and standards are added to appropriate wells along with the reaction mix.

After a brief incubation, around 10-15 minutes, the optical density of each well can be determined at a wavelength of 620 nm. Colorimetric kits offer a quick method of semi-quantifying zinc intracellularly at a relatively low cost.

Flow Cytometry

Flow cytometry may be preferred instead to measure intracellular zinc. Flow cytometry uses selective and membrane permeable fluorescent probes for specific detection and are appropriate for adherent cells and cells in suspension. These kits normally include a positive control that elevates zinc staining, and a cell- permeable zinc chelator that enables the separate detection of positive controls and the sample.

In experimentation, cell samples are first prepared by incubating them with an appropriate medium supplemented with 10% FBS at 37℃ in 5% CO2 overnight. After, the cells undergo pelleting, resuspension, and wash steps. Next, it is time to prepare controls; positive controls are generally supplied with the kit and require the addition of a zinc chelator, and negative controls are simply unstained cells. The controls and samples are then incubated, harvested, washed, and resuspended in an assay buffer. Next, the zinc fluorescent probe is supplied to each well followed by another incubation. After wash and resuspension steps, the plate can be analyzed using a flow cytometry at an Ex/Em of 485/525 nm.

The plate can also be viewed under a fluorescence microscope, where zinc molecules, nuclei, and other cellular compartments will appear differently colored. Flow cytometry methods provide a simple robust quantitative system of zinc measurement, and do not require the preparation of a standard curve.

Fluorimetric

Fluorescent detection kits for other biological materials, like serum, plasma, or urine, are also commercially available for the intracellular measurement of zinc. Similar to colorimetry, serial dilutions of the standard curve must first be created to aid with downstream analysis. Next, samples are prepared which may require neutralization and/or deproteinization. Samples and standards are then added to the test plate along with the fluorescent zinc probe reagent and assay buffer. The plate is then incubated, typical at room temperature for 5-10 minutes, and then the fluorescent intensity can be measured using a microplate reading at an Ex/Em of 485/525 nm.

Fluorescent kits provide a powerful means of semi-quantitatively measuring intracellular zinc, have little responses to other cations present in the sample, and produce a higher sensitivity over colorimetry, with a detection sensitivity of 0.2 µM or lower.

Tools:

Serial Dilution Calculator and Planner

Other Methods of Intracellular Zinc Measurement

---

A number of other techniques have been used throughout the literature to determine intracellular zinc levels. Importantly, förster resonance energy transfer (FRET) probes and sensors have been developed to provide accurate quantitative measurements. One emerging technology includes the use of small molecule probes that increase in fluorescence upon chelation to Zn2+. The strengths of small molecule probes lie in that they can be made cell permeable, yield a high fluorescence signal over potential background autofluorescence, and can be adjusted to fluoresce at various wavelengths. Alternatively, FRET-based protein sensors consist of two fluorescent proteins (an acceptor and donor) and a Zn2+ coordinating site. The ratiometric nature of these sensors allows for intracellular zinc quantitation that can be corrected to account for protein concentration and sample thickness. FRET-based protein sensors have the ability to target organelles and can be fine-tuned to bind Zn2+ with multiple affinities. These sensors have been derived from many distinctly colored fluorescent proteins, increasing their flexibility and use in research.



Not to be understated, a number of other types of sensors have been developed for measuring intracellular zinc. Hybrid and chemogenetic sensors combine synthetic small-molecule fluorescent sensors with a genetically encoded component to facilitate the identification and localization of zinc. Such sensors can brightly detect and effectively target zinc in samples. Alternatively, DNAzyme probes can detect metal ions using organic fluorophores or fluorescent proteins. These sensors are activated through exposure to light and then cleave in the presence of Zn2+. Bioluminescence resonance energy transfer (BRET) technology has also been used to create sensors that not only can image zinc fluxes in vivo by combining ratiometry with the action of luciferase to produce light upon interaction with zinc. In research, BRET-based sensors have measured intracellular zinc with lower background interference and less heterogeneity than fluorescent techniques.

Mass spectrometry-based techniques have also been used to assess intracellular zinc and involve the identification of specific chemicals within a sample through the separation of ions depending on their mass-to-charge ratio. Some approaches couple mapping capabilities that enable researchers to define the spatial distribution of metal ions, like zinc, within a sample. Generally, mass spectrometry techniques fall into two categories:

1. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is widely applied in life sciences for analyzing biological samples for metal localization and has been used to quantify Zn2+ in tumor growth as well as assess its role in metabolism and disease.
2. Secondary ion mass spectrometry (SIMS) uses a focused primary ion beam to dispel particles from a sample surface with the combined analysis in a mass spectrometer. Though this technique is less used in biology, it has the potential to detect lighter metals outside of just zinc, like potassium and silicon, which cannot be examined as successfully by other means.

X-Ray Fluorescence is another approach for mapping the distribution of metallic ions in biological specimens and can provide increasingly useful elemental and chemical information. Synchrotron x-ray fluorescence utilizes high-intensity photons as a power source which provides high sensitivity and submicron spatial resolution. This method is generally non-destructive, and the sensitivity of the technique increases with atomic number making it well-suited to study trace elements and metalloids, like zinc.

A subset of the technique, x-ray absorption spectroscopy, can be used to analyze the chemical speciation of individual elements. In this way researchers cannot only map zinc distribution within a sample but also obtain a detailed analysis of specialized regions within the sample. A third x-ray technique, electron-probe energy-dispersive spectroscopy (EDS), also electron probe microanalysis (EPMA), is like the aforementioned technique but instead uses an electron beam as the power source. It is usually used alongside a scanning and/or transmission electron microscope (SEM, TEM, STEM) to analyze specific morphological features of a starting material. In research, it has been successfully used to study the distribution of metals within a sample, including zinc, iron and potassium.

Application Notes:

Advances in two photon scanning and scanless microscopy technologies for functional neural circuit imaging

Product Ordering Information

---

References

---

The role of intracellular zinc release in aging, oxidative stress, and Alzheimer's disease
Role of intracellular zinc in molecular and cellular function in allergic inflammatory diseases
Techniques for measuring cellular zinc
The emerging role of zinc transporters in cellular homeostasis and cancer
Tools and techniques for illuminating the cell biology of zinc