This work was supported by the Faculty Diversity Scholars Award from the University of Massachusetts Medical School to T.P.-B. N.N.-T. is supported by SEP-CONACYT, grant 279879. J.G.N is supported by the National Science Foundation Grant DBI 0959476. The authors are grateful to Dr. Daryl A. Bosco for providing the N2A cell line and to Daniella Cangussu for her technical support.

1. Mammalian cell culture
<ol>
	<li>General considerations
	<ol>
		<li>Follow the aseptic techniques for mammalian cell culture previously reviewed38.</li>
		<li>Maintain all cell lines in a humidified 5% CO2 incubator at 37 &#176;C. Cell culture conditions vary for each cell type. It is important to maintain appropriate culture conditions for each cell line used, as variations of these procedures will lead to aberrant phenotypes and failure of the cell culture.</li>
...

Atomic absorbance spectroscopy is a highly sensitive method for Zn quantification in small volume/mass biological samples. The described optimization of Zn measurement makes application of this method simple and guarantees ideal analytical conditions. Here, using GF-AAS, we determined the concentration of Zn in whole cells, and in cytosolic and nuclear fractions, from different cell lines. The results show that this technique renders comparable to those obtained by fluorescent probes and ICP-MS. For instance, several flu...

Transition and heavy metals, such as Zn, Cu, Mn, and Fe, are found naturally in the environment in both nutrients in food and pollutants. All living organisms require different amounts of these micronutrients; however, exposure to high levels is deleterious to organisms. Metal acquisition is mainly through the diet, but metals can also be inhaled or absorbed through the skin1,2,3,4,5. It is important to note that the presence of metals in atmospheric particles is increasing and has been largely associated with health risks. Due to anthropogenic activities, increased levels of heavy metals such as Ag, As, Cd, Cr, Hg, Ni, Fe, and Pb have been detected in atmospheric particulate matter, rainwater, and soil6,7. These metals have the potential to compete with essential physiological trace elements, particularly Zn and Fe, and they induce toxic effects by inactivating fundamental enzymes for biological processes.
The trace element Zn is redox neutral and behaves as a Lewis acid in biological reactions, which makes it a fundamental cofactor necessary for protein folding and catalytic activity in over 10% of mammalian proteins8,9,10; consequently, it is essential for diverse physiological functions8,11. However, like many trace elements, there is a delicate balance between these metals facilitating normal physiological function and causing toxicity. In mammals, Zn deficiencies lead to anemia, growth retardation, hypogonadism, skin abnormalities, diarrhea, alopecia, taste disorders, chronic inflammation, and impaired immune and neurological functions11,12,13,14,15,16,17,18. In excess, Zn is cytotoxic and impairs absorption of other essential metals such as copper19,20,21.
Additionally, some metals like Cu and Fe have the potential to participate in harmful reactions. Production of reactive oxygen species (ROS) via Fenton chemistry can interfere with the assembly of iron sulfur cluster proteins and alter lipid metabolism22,23,24. To prevent this damage, cells utilize metal-binding chaperones and transporters to prevent toxic effects. Undoubtedly, metal homeostasis must be tightly controlled to ensure that specific cell types maintain proper levels of metals. For this reason, there is a significant need to advance techniques for accurate measurement of trace metals in biological samples. In developing and mature organisms there exists a differential biological need for trace elements at the cellular level, at different developmental stages, and in normal and pathological conditions. Therefore, precise determination of tissue and systemic metal levels is necessary to understand organismal metal homeostasis.
Graphite furnace atomic absorption spectrometry (GF-AAS) is a highly sensitive technique used for small sample volumes, making it ideal to measure transition and heavy metals present in biological and environmental samples25,26,27,28. Moreover, due to high sensitivity of the technique, it has been shown to be appropriate for studying the fine transport properties of Na+/K+-ATPase and gastric H+/K+-ATPase using Xenopus oocytes as a model system29. In GF-AAS, the atomized elements within a sample absorb a wavelength of radiation emitted by a light source containing the metal of interest, with the absorbed radiation proportional to the concentration of the element. Elemental electronic excitation takes place upon absorption of ultraviolet or visible radiation in a quantized process unique for each chemical element. In a single electron process, the absorption of a photon involves an electron moving from a lower energy level to higher level within the atom and GF-AAS determines the amount of photons absorbed by the sample, which is proportional to the number of radiation absorbing elements atomized in the graphite tube.
The selectivity of this technique relies on the electronic structure of the atoms, in which each element has a specific absorption/emission spectral line. In the case of Zn, the absorbance wavelength is 213.9 nm and can be precisely distinguished from other metals. Overall, GF-AAS can be used to quantify Zn with adequate limits of detection (LOD) and high sensitivity and selectivity25. The changes in absorbed wavelength are integrated and presented as peaks of energy absorption at specific and isolated wavelengths. The concentration of the Zn in a given sample is usually calculated from a standard curve of known concentrations according to the Beer-Lambert law, in which the absorbance is directly proportional to the concentration of Zn in the sample. However, applying the Beer-Lambert equation to GF-AAS analyses also presents some complications. For instance, variations in the atomization and/or non-homogenous concentrations of the samples can affect the metal measurements.
The metal atomization required for GF AAS trace elemental analysis consists of three fundamental steps. The first step is desolvation, where the liquid solvent is evaporated; leaving dry compounds after the furnace reaches a temperature of around 100 &#176;C. Then, the compounds are vaporized by heating them from 800 to 1,400 &#176;C (depending on the element to be analyzed) and become a gas. Finally, the compounds in the gaseous state are atomized with temperatures that range from 1,500 to 2,500 &#176;C. As discussed above, increasing concentrations of a metal of interest will render proportional increases on the absorption detected by the GF-AAS, yet the furnace reduces the dynamic range of analysis, which is the working range of concentrations that can be determined by the instrument. Thus, the technique requires low concentrations and a careful determination of the dynamic range of the method by determining the LOD and limit of linearity (LOL) of the Beer-Lambert law. The LOD is the minimum quantity required for a substance to be detected, defined as three times the standard deviation of Zn in the matrix. The LOL is the maximum concentration that can be detected using Beer-Lambert law.
In this work, we describe a standard method to analyze the levels of Zn in whole cell extracts, cytoplasmic and nuclear fractions, and in proliferating and differentiating cultured cells (Figure 1). We adapted the rapid isolation of nuclei protocol to different cellular systems to prevent metal loss during sample preparation. The cellular models used were primary myoblasts derived from mouse satellite cells, murine neuroblastoma cells (N2A or Neuro2A), murine 3T3 L1 adipocytes, a human non-tumorigenic breast epithelial cell line (MCF10A), and epithelial Madin-Darby canine kidney (MDCK) cells. These cells were established from different lineages and are good models for investigating lineage specific variations of metal levels in vitro.
Primary myoblasts derived from mouse satellite cells constitute a well-suited in vitro model to investigate skeletal muscle differentiation. Proliferation of these cells is fast when cultured under high serum conditions. Differentiation into the muscular lineage is then induced by low serum conditions30. The murine neuroblastoma (N2A) established cell line was derived from the mouse neural crest. These cells present neuronal and amoeboid stem cell morphology. Upon differentiation stimulus, the N2A cells present several properties of neurons, such as neurofilaments. N2A cells are used to investigate Alzheimer&#39;s disease, neurite outgrowth, and neurotoxicity31,32,33. The 3T3-L1 murine pre-adipocytes established cell line is commonly used to investigate the metabolic and physiological changes associated with adipogenesis. These cells present a fibroblast-like morphology, but once stimulated for differentiation, they present enzymatic activation associated with lipid synthesis and triglycerides accumulation. This can be observed as morphological changes to produce cytoplasmic lipid droplets34,35. MCF10A is a non-tumor mammary epithelial cell line derived from a premenopausal woman with mammary fibrocystic disease36. It has been widely used for biochemical, molecular, and cellular studies related to mammary carcinogenesis such as proliferation, cell migration, and invasion. The Madin-Darby canine kidney (MDCK) epithelial cell line has been extensively used to investigate the properties and molecular events associated with the establishment of the epithelial phenotype. Upon reaching confluence, these cells become polarized and establish cell-cell adhesions, characteristics of mammalian epithelial tissues37.
To test the ability of AAS to measure the levels of Zn in mammalian cells, we analyzed whole and subcellular fractions (cytosol and nucleus) of these five cell lines. AAS measurements showed different concentrations of Zn in these cell types. Concentrations were lower in proliferating and differentiating primary myoblasts (4 to 7 nmol/mg of protein) and higher in the four established cell lines (ranging from 20 to 40 nmol/mg of protein). A small non-significant increase in Zn levels was detected in differentiating primary myoblasts and neuroblastoma cells when compared to proliferating cells. The opposite effect was detected in differentiated adipocytes. However, proliferating 3T3-L1 cells exhibited higher concentrations of the metal compared to differentiated cells. Importantly, in these three cell lines, subcellular fractionation showed that Zn is differentially distributed in the cytosol and nucleus according to the metabolic state of these cells. For instance, in proliferating myoblasts, N2A cells, and 3T3-L1 pre-adipocytes, a majority of the metal is localized to the nucleus. Upon induction of differentiation using specific cell treatments, Zn localized to the cytosol in these three cell types. Interestingly, both epithelial cell lines showed higher levels of Zn during proliferation compared to when reaching confluence, in which a characteristic tight monolayer was formed. In proliferating epithelial cells, the mammary cell line MCF10A had an equal Zn distribution between the cytosol and nucleus, while in the kidney-derived cell line, most of the metal was located in the nucleus. In these two cell types, when the cells reached confluence, Zn was predominantly located to the cytosol. These results demonstrate that GF-AAS is a highly sensitive and accurate technique for performing elemental analysis in low-yield samples. GF-AAS coupled with subcellular fractionation and can be adapted to investigate the levels of trace metal elements in different cell lines and tissues.

<table>
	<tbody>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma Aldrich</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>1005706</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Brg1-antibody (G7)</td>
			<td>Santa Cruz biotechnologies</td>
			<td>sc-17796</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti b-tubulin-antibody (BT7R)</td>
			<td>Thermo Scientific</td>
			<td>MA5-16308</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford</td>
			<td>Biorad</td>
			<td>5000205</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Media (DMEM)</td>
			<td>ThermoFischer-Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Media/Nutrient Mix (DMEM/F12)</td>
			<td>ThermoFischer-Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>ThermoFischer-Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidemal Growth Factor (EGF)</td>
			<td>Sigma Aldrich</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFischer-Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblastic Growth Factor-Basic (FGF) (AA 10-155)</td>
			<td>ThermoFischer-Gibco</td>
			<td>PHG0024</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>ThermoFischer-Gibco</td>
			<td>16050122</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma Aldrich</td>
			<td>H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (H2O2)</td>
			<td>Sigma Aldrich</td>
			<td>95321</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>91077C</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-A</td>
			<td>ThermoFischer</td>
			<td>51300044</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric Acid (HNO3)</td>
			<td>Sigma Aldrich</td>
			<td>438073</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40 (NP-40)</td>
			<td>Thermo Scientific</td>
			<td>85125</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM (Reduced Serum Media)</td>
			<td>ThermoFischer-Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>ThermoFischer-Gibco</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>PureCol (Collagen)</td>
			<td>Advanced BioMatrix</td>
			<td>5005</td>
			<td></td>
		</tr>
		<tr>
			<td>Retionic Acid</td>
			<td>Sigma Aldrich</td>
			<td>PHR1187</td>
			<td></td>
		</tr>
		<tr>
			<td>Troglitazone</td>
			<td>Sigma Aldrich</td>
			<td>648469-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFischer-Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc (Zn) Pure Single-Element Standard, 1,000 &#181;g/mL, 2% HNO3</td>
			<td>Perkin Elmer</td>
			<td>N9300168</td>
			<td></td>
		</tr>
		<tr>
			<td>Established Cell Lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3T3-L1</td>
			<td>American Type Culture Collection</td>
			<td>CL-173</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDK</td>
			<td>American Type Culture Collection</td>
			<td>CCL-34</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF10A</td>
			<td>American Type Culture Collection</td>
			<td>CRL-10317</td>
			<td></td>
		</tr>
		<tr>
			<td>N2A</td>
			<td>American Type Culture Collection</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Absortion spectrophotometer</td>
			<td>PerkinElmer</td>
			<td>Aanalyst 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagnode</td>
			<td>UCD-200</td>
			<td></td>
		</tr>
	</tbody>
</table>

atomic absorbance spectroscopy to measure intracellular zinc pools in mammalian cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

We tested the ability of the GF-AAS to detect minute levels of Zn in mammalian cells (Figure 1). Thus, we cultured primary myoblasts derived from mouse satellite cells, and the established cell lines N2A (neuroblastoma derived), 3T3 L1 (adipocytes), MCF10A (breast epithelium), and MDCK cells (dog kidney epithelium). First, we isolated whole cell, cytosolic, and nuclear fractions of all these cell types and evaluated the purity of the fractions by western blot...

Watch this Scientific Journal Video about Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells at JoVE.com

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

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38.3K visualizações.  University of Massachusetts Medical School. O papel dos metais de transição no desenvolvimento dos mamíferos é em grande parte subestimado. Evidências emergentes mostram que a homeostase metálica continua a mudar de acordo com o estágio de desenvolvimento das células e tecidos. Além disso, os destinos celulares específicos dos metais também variam em desenvolvimento, e, claro, é desafiador identificar sua localização.Até o momento, diferentes técnicas analíticas foram desenvolvidas para quantificar metais, mas a...