Name: atomic absorbance spectroscopy to measure intracellular zinc pools in mammalian cells
Uploaded: 2019-05-16T15:00:00
Description: Watch this Scientific Journal Video about Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells at JoVE.com

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>
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			<td>I5879</td>
			<td></td>
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			<td>Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>1005706</td>
			<td></td>
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			<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>
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			<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 ...

The role of transition metals in mammalian development is largely underestimated. Emerging evidence shows that metal homeostasis continues to change according to the developmental stage of cells and tissues. Moreover, the specific cellular destinations of metals also varietal in development, and of course, it's challenging to identify their location.To date, different analytical techniques have been developed to quantify metals, but most are expensive or inaccessible. Intensive research is focused to improve metal determinations in a more accessible and efficient manner. Graphite furnace atomic absorption spectroscopy, GF-AAS, has two main advantages.It provides low limits of detection for zinc, allowing for accurate trace zinc analysis. It is also a technique that does not require high volumes of sample, microliters of solution tend to be enough to carry out the analysis. The combination of these two advantages make GF-AAS ideal to analyze trace elements in low volume samples.Several human pathological conditions are related to metal misbalances. Some examples are anemia, acrodermatitis enteropathica, and Wilson's and Menkes'diseases. Therefore, it is important to develop efficient and reliable methods to measure the levels of transition metals in biological samples with high sensitivity and accuracy.GF-AAS is an excellent example on a technology that facilitates a determination of any given metal in normal physiological conditions, and in pathogenic cases. The method is very versatile. It can be applied to many systems and many elements can be analysis using GF-AAS.The challenges are to establish the method limit of detection in reality, making sure that the samples fall within this limit. Given the fact that the volume of samples is low, there is not a lot of room for testing, so carefully examination of data needs to take place since the very beginning. After the first analysis of samples, we need to establish what's the dilution factor needed to bring the samples to the limits required for quantification.Be sure that you are able to fractionate the nuclei properly before going to the AAS. Western blots are always good validation prior to the spectroscopic analysis. While GF-AAS requires low volumes, intracellular pools are in low volumes already.That gives little room for testing. The first measurements need to be carefully done and examined, so sample treatment can be minimized to just one dilution factor. To begin, culture the cells of interest in 55 square centimeter plates.Use a vacuum trap to aspirate the culture media. Be sure to remove all traces of media. Use ice cold PBS free of calcium and magnesium to rinse the cells from the desired time points, or culture conditions, three times.Then add one milliliter of ice cold PBS to the plate, and scrape the cells off the plate. Transfer the cell suspensions to a 1.5 milliliter microcentrifuge tube, and keep it on ice. Centrifuge for 10 seconds at 10, 000 times G, and remove the supernatant by aspiration.If metal analysis of cytoplasmic and nuclear fractions is desired, resuspend the cell pellet in 400 microliters of ice cold PBS containing 0.1%NP-40, a non-ionic detergent. Transfer 100 microliters to a new microcentrifuge tube as the whole cell sample, and store it at minus 20 degrees celsius. To isolate nuclei, use a P1000 micropipette tip to pipette the 400 microliter cell suspension up and down five to 10 times on ice.Then, extract five microliters of the cell suspension and transfer it to a microscope glass slide. Place the five microliters under a light microscope using a 40 times objective to verify the nuclei integrity. Centrifuge the remaining 395 microliter cell lysate suspension for 10 seconds at 10, 000 times G.Transfer the supernatant containing the cytosolic fraction to a new microcentrifuge tube.Next, add 500 microliters of the PBS containing 0.1%NP-40 to rinse the pellet with the nuclear fraction, and centrifuge for 10 seconds at 10, 000 times G.Remove the supernatant, and resuspend the pellet containing the nuclei in 100 microliters of the same solution. To lyse the cells, use a Bioruptor to sonicate the whole cell and nuclei containing solution three times, each for five minutes. Proceed to perform quality control of the purity of the fractions by western blot, using antibodies specific for either the nuclear or cytosolic fractions.First, add an equal volume of concentrated nitric acid of trace metal grade to the whole cell, cytoplasmic and nuclear fractions containing 100, 500 and 100 microliters of cell suspension in 1.5 milliliter microcentrifuge tubes, and place them in a thermal block at 80 degrees celsius to mineralize the cell culture for one hour. After that, take out the tubes from the thermal block and place them in a rack to continue the acid digestion overnight at 20 degrees celsius. Stop the reaction by adding 30%of the sample volume of hydrogen peroxide.Bring the sample volume to 500 microliters with 18 mega Ohm purified water. Next, in a 15 milliliter Falcon tube dilute analytical grade standard nitric oxide in purified water at 18 mega Ohms to prepare two volume percent nitric acid solution as the blank solution during calibration. Then, use the two volume percent nitric acid solution to dilute a commercially available 1, 000 ppm zinc stock solution with a concentration of 1, 000 parts per billion.Prepare working standard solutions from the 1, 000 part per million zinc standard solution, the 1, 000 part per billion solution, then dilute it to 5, 8, 10, 15, 20, and 25 ppb directly with 2 volume percent nitric acid solution. Place one milliliter of 0.1%magnesium nitrate matrix modifier into polypropylene sample cups, and then load the cups into the autosampler carousel. Program the atomic absorption spectrometer to automatically add five microliters of the matrix modifier to the zinc standards and the blank.Set the same optimized condition on the atomic absorption spectrometer for the standard solutions and the samples, with introduction flow rate at 250 milliliters per minute, injection temperature at 20 degrees celsius, and graphite furnace temperature reaching 1, 800 degrees celsius in four step processes. All the standards and samples of a single experiment should be measured at the same time to avoid mistakes due to calibration differences or evaporation. Optimize the lamp C-HCL to a current of 20 amps, wavelength of 213.9 nanometers, and slit of 0.7 nanometers.Pipette the samples in polypropylene sample cups, and place them into the autosampler carousel. The lower limit of detection of zinc is 0.01 parts per billion. By increasing the concentration of the standards, the upper limit of detection of zinc is determined to be 20 parts per billion.After a zinc standard curve is obtained, measure the mineralized samples to determine metal content. If the data obtained is beyond the limit of detection, dilute the samples with 18 mega Ohm purified water treated with 0.1%analytical grade nitric acid. In this rapid isolation of nuclei protocol, a representative western blot from differentiating primary myoblasts shows the purity of subcellular fractions.The chromatin remodeler enzyme Brg1 was used to identify the nuclear fraction, and tubulin was used to identify the cytosolic fraction. This figure shows representative light micrographs for proliferating and differentiated or confluent monolayers of each cell type, and the corresponding zinc content in whole cell extracts, cytosolic and nuclear fractions. All the cell lines analyzed in this study showed zinc concentrations in the nanomolar range.Differentiated primary myotubes exhibited higher levels of zinc than proliferating cells. A similar subcellular distribution of zinc was detected in the neuroblastoma derived cell line N2A. On the other hand, the established 3T3-L1 cell line exhibited higher levels of zinc when the pre-adipocytes were proliferating than when they were induced or differentiated.MCF10A cells showed equal levels of zinc between cytosolic and nuclear fractions in proliferating cells. Once MCF10A cells reach confluence, a 40%decrease in whole cell zinc levels was detected, and the metal was found to be most concentrated in the cytosolic fraction. Graphite furnace atomic absorption spectrometry is a highly sensitive technique to measure transition and heavy metals in biological and environmental samples.Special care and cleanliness in the preparation of subcellular fractions should be taken, as minimal contamination will likely interfere with the analysis, due to the sensitivity of the equipment. Given the low volumes and trace levels of metals in every experiment, it is difficult to think of a better and more accurate technique to measure zinc than GF-AAS. There are no current techniques that can provide the limits of detection and low volume required at the relatively low cost of GF-AAS.Graphite furnace atomic absorption spectrometry is accurate, sensitive, cost-effective, and accessible. This analytical technique will continue to improve as elemental detection technologies continues to advance. Future application for detection of zinc and other metals by GF-AAS will include organs, tissues obtained from animal models, and biopsies from patients with disease associated with systemic metal imbalances.Nitric acid is an extremely corrosive acid capable of causing severe chemical burns very rapidly. This chemical can also react violently with certain compounds such as metallic powders. Because of the hazard posed by nitric acid, it's important to take strict safety measures whenever handling nitric acid.When handling nitric acid we strongly recommend you wear safety chemical glasses, face shield for splash protection, gloves, and approved vapor respiration if ventilation is not adequate.

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