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>
		<li>Ensure acquaintance with the cell line of interest, and follow the protocols provided for each cell type selected for specific experiments (see some examples below).</li>
	</ol>
	</li>
	<li>General procedure for trypsinization and passing of adherent cells
	<ol>
		<li>Remove the cell culture media from the culture plate by aspiration.</li>
		<li>Wash the cells gently using PBS without calcium and magnesium (5 mL per 10 cm2 culture surface area). Add the wash solution to the side of the plate to avoid detaching cell monolayer; swirl the plate to maximize coverage of the solution. For epithelial cells, it is necessary to incubate the cells for 10 to 15 min in the incubator at 37 &#176;C in PBS to facilitate the dissociation of cells in the following steps.</li>
		<li>Remove the washing solution by aspiration.</li>
		<li>Add enough dissociation reagent (0.25% trypsin) to the plate (approximately 0.7 mL per 10 cm2). Gently swirl the plate to get complete coverage of the cell monolayer.</li>
		<li>Incubate the culture at room temperature for 2 to 5 min. The actual incubation time varies with the cell line used, in the case of epithelial cells is recommended to incubate for 10 to 15 min in the incubator at 37 &#176;C. Observe the cells under the microscope for cell detachment.</li>
		<li>When most of the cells have detached, recover the cell suspension in 5 mL of the pre-warmed plating or growth medium (see below for some examples). Mechanically dissociate cell clumps by pipetting over the cell monolayer several times.</li>
		<li>Transfer the cell suspension to a 15 mL conical tube and centrifuge them at 1,000 x g for 2 to 5 min. The centrifugation speed and time vary based on the cell type. Remove the media by gentle aspiration, being careful not to touch the cell pellet. Resuspend the cell pellet in 5 to 10 mL of the appropriate pre-warmed growth medium and remove a sample for counting using a hemocytometer or an automatic cell counter.</li>
		<li>Utilize the appropriate volume of the cell suspension to seed the recommended density for each specific cell line (see below for some examples). Supplement with the adequate volume of culture media according to the size of the culture dish and place the cells back into the incubator.</li>
	</ol>
	</li>
	<li>Primary myoblasts derived from mouse satellite cells
	<ol>
		<li>Before starting, prepare collagen-coated plates for primary myoblast growth. A solution containing 0.02% collagen, and 0.05 M acetic acid in deionized water must be sterilized by filtration with a 0.22 &#181;m sterile filter device. Incubate the culture plates with the collagen solution overnight at 37 &#176;C.</li>
		<li>On the next day, aspirate the collagen solution, allow the plates to air dry in the sterile cabinet and store at 4 &#176;C until use. 
		NOTE: The minimal volumes of collagen are: for 35 mm = 1 mL; 60 mm = 2 mL; 10 cm = 5 mL.</li>
		<li>Seed primary myoblasts at 1 x 104 cells/cm2 in 55 cm2 collagen-coated culture plates. The proliferation media is Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/Ham&#8217;s F12 nutrient mixture culture media containing 20% fetal bovine serum (FBS), 25 ng/mL of recombinant basic fibroblastic growth factor (FGF), and 100 U/mL of penicillin/streptomycin. 
		NOTE: Proliferating and stock cells should be maintained under 50% of confluency. Cell contact induces commitment of the cells to myogenic differentiation and impairs the growth capabilities of the culture.</li>
		<li>For differentiating the primary myoblasts, allow the cells to reach 80% to 100% confluency, which normally occurs after 48 h of plating. Then, change to differentiation media (DMEM, 2% horse serum, 1% insulin, transferrin, selenium A supplement, and 100 U/mL of penicillin/streptomycin). Refresh the media daily until harvest.</li>
	</ol>
	</li>
	<li>Murine neuroblastoma (N2A)
	<ol>
		<li>Plate N2A cells at a density of 2 x 104 cells/cm2 in growth media, containing a mixture of reduced-serum medium/DMEM containing high glucose and L-glutamine (1:1, v:v), supplement with 10% FBS and 100 U/mL of penicillin/streptomycin. Maintain the stock culture under 50% confluency.</li>
		<li>Induce differentiation of N2A cells once confluency reached 25% to 40%. Change to differentiation media containing a mixture of reduced-serum medium/DMEM, 1% FBS, and 20 &#181;M retinoic acid for 7 to 10 days. Refresh the media every other day until cell harvest. 
		NOTE: As N2A cells differentiate, some cells become round, detach easily, and may undergo apoptosis. Be careful when aspirating and adding the culture media to the plates.</li>
	</ol>
	</li>
	<li>3T3 L1 murine pre-adipocytes
	<ol>
		<li>Maintain mouse 3T3/L1 cells in DMEM with high glucose, supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin. The adipogenic process is dependent in the confluence of the cells; therefore, do not let the stock culture grow over 50% confluency, as higher confluence will impair the ability of the cells to differentiate.</li>
		<li>Plate the cells at 2 x 104 cells/cm2. At this density, the cells will reach confluence in 3 days.</li>
		<li>Induce adipogenic differentiation two days after the cells reach 100% confluency. Induction media consists of DMEM containing 10% FBS, 10 &#956;g/mL insulin, 0.5 mM 3-isobutyl-1-methyxanthine, 1 &#956;M dexamethasone, and 10 &#956;M troglitazone.</li>
		<li>Replace the induction media after 48 h incubation to differentiation media (DMEM containing 10% FBS, and 5 &#956;g/mL insulin). Refresh the media every other day until harvest. Representative samples of full differentiation are normally collected between 7 and 10 days after adipogenic induction. 
		NOTE: As adipogenesis progresses, the cells become round and tend to detach easily. Be careful when aspirating and adding the culture media to the plates.</li>
	</ol>
	</li>
	<li>MCF10A cells
	<ol>
		<li>Plate MCF10A cells in DMEM/F12 (1:1, v:v), supplemented with 5% FBS, 100 U/mL penicillin/streptomycin, 0.5 &#181;g/mL hydrocortisone, 10 &#181;g/mL insulin, and 20 ng/mL epidermal growth factor (EGF). Recommended seeding density is 1 x 105 cells/cm2. Under these conditions, the cells will reach 100% confluency within 4 days. Refresh the media every 2 to 3 days until harvest. 
		NOTE: MCF10A cells are difficult to detach. It is recommended to incubate the cells for 10 to 15 min at 37 &#176;C in PBS free of calcium with 0.5 mM EDTA prior to trypsinization.</li>
	</ol>
	</li>
	<li>Madin-Darby canine kidney cells (MDCK)
	<ol>
		<li>Plate MDCK cells in DMEM supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin. Recommended seeding density is 1 x 105 cells/cm2, where the cells will reach 100% confluency within 3 days. Refresh the media every 2 days until harvest. 
		NOTE: MDCK cells are difficult to detach. It is recommended to incubate the cells for 5 to 10 min at 37 &#176;C in PBS free of calcium and magnesium prior to trypsinization. To prevent cell clumping do not agitate the plates by hitting or shaking the flask while waiting for the cells to detach.</li>
	</ol>
	</li>
</ol>
2. Mammalian cell culture sample preparation for AAS: whole cell and subcellular fractionation
<ol>
	<li>Culture the cells of interest in 55 cm2 plates. The use of smaller culture plates may yield samples with levels of metals under the detection limits of the GF-AAS.</li>
	<li>Whole cell extract
	<ol>
		<li>Aspirate the culture media using a vacuum trap. Be sure to remove all traces of media. Rinse the cells from the desired time points or culture conditions three times with ice-cold PBS free of calcium and magnesium.</li>
		<li>Immediately scrape the cells from the plate using a new plastic cell scraper in 1 mL of PBS free of calcium and magnesium. Transfer the sample to a 1.5 mL microcentrifuge tube and centrifuge for 2 min at 2,000 x g and aspirate the supernatant. The protocol can be paused here. Store pellet samples at -20 &#176;C or proceed to step 2.4.</li>
	</ol>
	</li>
	<li>Subcellular fractionation
	<ol>
		<li>Aspirate the culture media using a vacuum trap. Be sure to remove all traces of media. Rinse the cells from the desired time points or culture conditions three times with ice-cold PBS free of calcium and magnesium. Scrape the cells off the plate with 1 mL of ice-cold PBS free of calcium and magnesium.</li>
		<li>Transfer the samples to a 1.5 mL microcentrifuge tube and keep on ice. Centrifuge for 10 s at 10,000 x g. Continue rapid isolation of nuclei if metal analysis of cytoplasmic and nuclear fractions is desired. This procedure will minimize the potential loss of metals due to cell extraction39,40.</li>
		<li>Remove the supernatant by aspiration and resuspend the cell pellet in 400 &#956;L of ice-cold PBS free of calcium and magnesium, containing 0.1% Nonidet P-40 (NP-40), a non-ionic detergent. Transfer 100 &#956;L to a new microcentrifuge tube and consider it as the whole cell sample. This aliquot represents 25% of the sample.</li>
		<li>Isolate nuclei by pipetting the cell suspension 5 to 10 times on ice with a P1000 micropipette tip. Nuclei isolation of epithelial cells requires a concentration of 0.5 % NP-40. Achieve cell dissociation by passing the cell suspension through a 26 &#190; G needle 10 to 15 times, followed by 10 to 15 passages through a 30 &#189; G needle. 
		NOTE: Verify the nuclei integrity by light microscopy using a 40x objective.</li>
		<li>Centrifuge the remaining cell lysate suspension (300 &#956;L) for 10 s at 10,000 x g. The supernatant will contain the cytosolic fraction. Transfer the 300 &#956;L to a new microcentrifuge tube.</li>
		<li>Rinse the pellet containing the nuclear fraction in 500 &#956;L PBS free of calcium and magnesium, containing 0.1% NP-40, then centrifuge for 10 s at 10,000 x g. Remove the supernatant and resuspend the pellet containing the nuclei in 100 &#956;L of the same solution. 
		NOTE: The protocol can be paused here. Samples can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Lyse the cells by three sonication cycles (30 s on/30 s off, at 50 kHz, 200 W) for 5 min. Quantify total protein content in the sample by Bradford41.</li>
	<li>Perform quality control of the purity of the fractions by western blot using antibodies specific for either the nuclear (histones, chromatin remodelers, nuclear matrix proteins) or cytosolic fractions (&#946;-tubulin, &#946;-actin) (Figure 2). 
	NOTE: Do not use sonicators with a metal tip. These can contaminate the sample with metals.</li>
</ol>
3. Zn content analysis of whole cell and subcellular fractions of mammalian cell cultures by atomic absorbance spectroscopy
<ol>
	<li>Mineralize the cell culture samples (whole cell or nuclear fractions) by acid digestion using an equal volume of concentrated HNO3 trace metal grade (CAUTION) for 1 h at 80 &#176;C, then overnight at 20 &#176;C.</li>
	<li>Stop the reaction by adding 30% of the reaction volume of H2O2. Bring the sample volume to 500 &#956;L with 18 M&#937; purified water. 
	NOTE: HNO3 handling should be done wearing chemical safety glasses, a face shield for splash protection, gloves, and an approved vapor respirator if adequate ventilation (fume hood) is not available. The protocol can be paused here. Samples can be stored at room temperature (RT).</li>
	<li>Prepare standard solutions and experimental samples for Zn measurements by AAS equipped with a graphite furnace.
	<ol>
		<li>Use 2% (v/v) HNO3 solution in deionized water, coming from the same batch used for the standard curve as the blank solution for calibration throughout. Use analytical grade standards diluted in 18 M&#937; purified water.</li>
		<li>Prepare a standard of 1,000 ppb of Zn from a commercially available 1,000 ppm Zn stock solution with 2% (v/v) HNO3 solution in deionized water. Prepare working standard solutions from the 100 ppb standard by diluting it to 5, 8, 10, 15, 20, and 25 ppb directly with 2% (v/v) HNO3 solution in deionized water. Prepare the Zn standards and the blank with a 0.1% (1 g/L) Mg(NO3)2 matrix modifier. 
		NOTE: The LOD of Zn is 0.01 ppb (&#181;g/L). By increasing the concentration of the standards, the limit of linearity of Zn was determined to be 20 ppb (Figure 3).</li>
		<li>Measure standard and samples under the same optimized conditions: flow rate of introduction of 250 mL/min, injection temperature of 20 &#176;C, and GF temperature of 1,800 &#176;C. 
		NOTE: The GF temperature was increased to 2450 &#176;C for cleanout steps, prior to the injection of a new sample or standard.</li>
		<li>Optimize the lamp (C-HCL) to a current of 20 A, wavelength of 213.9 nm, and slit of 0.7 nm.</li>
		<li>Measure the mineralized samples using the Zn standard curve to determine metal content. As small volumes are measured, the samples can evaporate if the measurements take long periods of time. Therefore, it is recommended to add water to the samples. Dilute the samples with 18 M&#937; purified water treated with 0.01% HNO3 (analytical grade) if the data obtained is beyond the LOD. 
		NOTE: Typically, samples are diluted to a volume of 500 &#181;L, placed in the automated autosampler of the AAS to be measured right after the standard curve is established. The required volume should be determined by the user and should consider final calculations of concentrations. Finishing the Zn determinations by AAS of a complete experiment in one attempt is recommended. Pausing the measurements may lead to large experimental variations and inconsistencies.</li>
	</ol>
	</li>
	<li>Convert the ppb measurements to molarity as obtained from Zn measured via AAS. Consider the mass of Zn &#8206;(65.39 amu). Divide the amount of ppb obtained by AAS by 63,390,000. In cells, the Zn concentrations range from units of pmol to &#181;mol.
	<ol>
		<li>Divide the concentration of Zn (pmol or &#181;mol) by the initial mass of protein in the sample determined by Bradford (step 2.4). This calculation will render the amount of metal (pmol or &#181;mol Zn) contained per mg of protein of the sample. 
		NOTE: It is important to consider the dilution factors utilized during the metal determinations.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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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Atomic Spectroscopy

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38.4K Views.  University of Massachusetts Medical School. 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.

Video: Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

Stable Isotope Tracing &amp; LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...