This work was funded by the NIH National Heart, Lung, and Blood Institute grant RO1 HL81248.

1. Culture of Bovine Aortic Endothelial Cells (BAEC)
<ol>
 <li>Proper aseptic techniques were followed.</li>
 <li>In a water bath, warm medium without antibiotics at 37 &deg;C.</li>
</ol>
Note: The medium consists of phenol free Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L D-glucose, 4 mM L-glutamine, 1% non-essential amino acids, supplemented with 10% fetal bovine serum (FBS) and 2.5 mg/L endothelial growth factor.
<ol start="3">
 <li>Remove the T75 flask containing cells from the incubator and clean the surface of the flask with 70% ethanol before placing it inside the hood. </li>
 <li>Remove the old medium using an aspirator and wash twice with 5 ml of Dulbecco's phosphate buffer saline (DPBS). </li>
 <li>Add 2 ml of trypsin and wait for 4-5 min for cells to detach while periodically inspecting under the microscope. </li>
 <li>Add 3 ml of the medium and repeatedly mix using a pipette to separate the cells and create an even suspension. </li>
 <li>Transfer 5 ml of the medium with trypsin to a 15 ml tube and centrifuge at 121 g for 5 min. </li>
 <li>Remove the supernatant using an aspirator. Add 5 ml of DPBS and mix thoroughly. Centrifuge at 121g for 5 min. </li>
 <li>Aspirate the supernatant and re-suspend the cell pellet by adding 6 ml of the medium. </li>
 <li>On a 6-well plate, add 1 ml of the cell suspension to each well then add 1 ml of the medium. Mix the suspension using a pipette. </li>
 <li>Label the plate and incubate overnight at 37 &deg;C and 5% CO2. </li>
</ol>
2A. Detection of NO with BAEC Cell
<ol>
 <li>Proper aseptic techniques were followed. </li>
 <li>Remove the 6-well plate from the incubator and aspirate the medium from the first well. Wash the cells twice with 1 ml DPBS. </li>
 <li>Add 210 &mu;l of 1.9 mM iron(II)sulfate heptahydrate (FeSO4.7H2O, prepare freshly by dissolving 0.8 mg in 1 ml DPBS with CaCl2 and MgCl2) and 210 &mu;l of ammonium N-methyl-D-glucamine dithiocarbamate (MGD, prepare freshly by dissolving 2.7 mg in 500 &mu;l DPBS with CaCl2 and MgCl2) using a ratio of 1:7. (Note: This ratio where excess MGD is used has been conventionally employed for the preparation of Fe2+-MGD complex due to the fact that the yield for Fe3+-MGD is maximized in the presence of excess MGD. The addition of ascorbate in solution to stabilize the Fe2+-MGD is not necessary since the NO-Fe3+-MGD formed is endogenously reduced to the EPR detectable NO-Fe2+-MGD by ascorbate, hydroquinone, or cysteine with conversion efficiency of up to 99.9%.. The low spin diamagnetic state of Fe2+ allows the detection of NO using flat cell or capillary tube without the need of a low temperature device)15. </li>
 <li>Swirl the resulting suspension well and add 4.6 &mu;l of calcium ionophore (CaI) (prepared from a stock solution of 1.9 mM by dissolving 1 mg in 1 ml DMSO). </li>
 <li>Swirl the solution again and incubate at 37 &deg;C for 36 min in order to further allow reduction of NO-Fe3+-MGD to the EPR detectable NO-Fe2+-MGD. </li>
 <li>Collect the supernatant (425 &mu;l) in an Eppendorf tube and transfer to an EPR flat cell (or to a 50 &mu;l capillary tube).</li>
 <li>EPR acquisition parameters are: microwave frequency: 9.8 GHz; center field: 3427 G; modulation amplitude: 6.0 G; sweep width: 100 G; receiver gain: 1 x 105; microwave power: 10 mW; total number of scans: 121; sweep time: 10 s; and time constant: 20 ms. (Note: Since parameters will vary from one instrument and experimental conditions to another, therefore, only the center field, frequency and modulation amplitude are the most important parameters to consider.) </li>
 <li>Record the spectra at room temperature and the 2-D spectra were integrated to reduce the background noise and baseline corrected using Bruker WinEPR Data Processing software or other data processing software. For quantification of adduct formation, standard plots of concentration versus signal intensity (or area) can be constructed using an NO donor SNAP. </li>
</ol>
2B. eNOS Uncoupling Experiment 
<ol>
 <li>Remove the 6-well plate from the incubator and aspirate the medium from the second well and wash twice with 1 ml DPBS. </li>
 <li>A peroxynitrite donor, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1) was used to uncouple eNOS16. Add 100 &mu;l of 0.5 mM SIN-1 (Mr 206.6 g/mol, from 10 mM stock solution freshly prepared by dissolving 1 mg SIN-1 in 500 &mu;l of PBS with no Ca/Mg ions) and dilute to 2 ml with DPBS and 10% FBS. </li>
 <li>Incubate for 2 h at 37 &deg;C and 5% CO2. </li>
 <li>Remove the plate from incubator and wash twice with DPBS.</li>
 <li>Add 210 &mu;l of 2.8 mM FeSO4.7H2O and 210 &mu;l of 19.6 mM MGD freshly prepared according to the procedure mentioned above.</li>
 <li>Swirl the solution and add 4.6 &mu;l of 1.9 mM CaI. </li>
 <li>Swirl the solution again and incubate at 37 &deg;C for 36 min. </li>
 <li>Collect the supernatant (425 &mu;l) in an Eppendorf tube and transfer to an EPR flat cell. </li>
 <li>EPR acquisition parameters are: microwave frequency: 9.8 GHz; center field: 3427 G; modulation amplitude: 6 G; sweep width: 100 G; receiver gain: 1 x 105; microwave power: 10 mW; total number of scans: 121; sweep time: 10 s; and time constant: 20 ms. </li>
 <li>Record the spectra and the 2-D spectra were integrated to reduce the background noise and baseline corrected as mentioned above. </li>
</ol>
3. Detection of O2&bull;- from Polymorphonuclear Neutrophils (PMNs)
<ol>
 <li>Neutrophils were isolated from human blood sample as previously described17. </li>
 <li>Make a stock solution of 1 M DMPO* in PBS containing 0.1 mM diethylenetriamine-pentaacetic acid (DTPA). DMPO has a melting point of 25-29 &deg;C so it is more convenient to pipette liquid DMPO (density ~ 1.02 g/ml at 25 &deg;C) in to a glass vial (Note: do not use plastic vials for weighing since pure DMPO reacts with plastic). Frozen DMPO can be melted by running luke warm water on to the vial (Note: do not run hot water as DMPO may decompose).</li>
 <li>It is important to use high purity DMPO (&gt; 99%) since some of the commercially available spin traps contain paramagnetic impurities, and therefore, it is imperative to run EPR spectrum of just the DMPO solution alone (10 mM in this case). It is critical that no background signal is evident (see Figure 3A).</li>
 <li>Prepare stock solution (1 mg/ml) of phorbol-12-myristate-13-acetate (PMA) in DMSO. Make aliquots by diluting the solution to 10 &mu;g/ml in PBS. </li>
 <li>In a 1.5 ml Eppendorf tube, prepare a solution with a total volume of 0.6 ml by following the sequence of addition: ~106 cells per ml of PMN, D-glucose (1 mg/ml) and albumin (1 mg/ml), 10 mM DMPO and 0.2 &mu;g/ml of PMA. (Note: PMA is the radical activator and should be added last). </li>
 <li>Transfer the solution to an EPR flat cell. </li>
 <li>EPR acquisition parameters conditions are: microwave frequency: 9.8 GHz; center field: 3486 G; modulation amplitude: 0.5 G; sweep width: 100 G; receiver gain: 5 x 105; total number of scans: 10; sweep time: 30 s; microwave power: 20 mW; and time constant: 81 ms. For quantification of adduct formation, standard plots of concentration versus signal intensity (or area) can be constructed using the stable nitroxides such as TEMPO or 3-carboxylic acid-PROXYL. </li>
</ol>
* Important note on the use of DMPO: By using a flat cell, one can increase the cell density and thereby increasing the signal intensity of the spin adduct but the half-life of O2&bull;- adduct of DMPO is short (t1/2 ~ 1 min) which decomposes to DMPO-OH. DMPO can be substituted using the same concentrations of EMPO, BMPO or DEPMPO which are available commercially for increased adduct stability with t1/2 ~ 8 and 14 min, respectively.
4. Representative Results
Spin trapping of NO radical was performed using Fe2+-MGD. Figure 2A and 2B show no EPR signal from Fe2+-MGD or mixture of Fe2+-MGD with CaI, indicating that no NO background signal originates from these reagents. BAEC upon stimulation with CaI releases NO which reacts with Fe2+-MGD to form the spin adduct, NO-Fe2+-MGD, and shows a characteristic triplet signal with hyperfine splitting constant (hfsc) value of aN = 12.66 G and g-factor of g= 2.040. (Figure 2C). The hfsc value was determined using the WINSIM simulation program that can be downloaded from NIEHS EPR Software Database website. The experimental hfsc is consistent with the literature value of aN=12.70 G and g = 2.04118 for NO-Fe2+-MGD adduct. Similarly, the effect of SIN-1 on BAEC lowered the NO production due to eNOS uncoupling as shown in Figure 2D.
DMPO spin trap was used for O2&bull;- detection. DMPO alone did not give a signal as shown in the Figure 3A confirming that the spin trap is free from paramagnetic impurities. Figure 3B is the spectrum of DMPO and PMNs only, and similarly, there is no detectable signal suggesting that the DMPO does not cause activation of the enzyme NADPH oxidase. Figure 3C shows the observed EPR signal upon stimulation of PMN by PMA. The hfsc values for this signal were determined to be aN =14.71 G, a&beta;-H =11.40 G and a&gamma;-H =1.25 G, and are consistent with the literature values of aN =14.3 G, a&beta;-H =11.7 G and a&gamma;-H =1.3 G 19 for DMPO-O2H adduct.
<img src="/files/ftp_upload/2810/2810fig1.jpg" alt="Figure 1" /> 
Figure 1. Flow chart for the detection of radicals from neutrophils and BAEC using EPR spin trapping. (A) PMNs were mixed with DMPO and PMA, and the resulting mixture transferred to an EPR flat cell for data acquisition. (B) BAEC were grown on a plate, and washed with DPBS. The spin trap Fe(MGD)2 was added along with CaI. The solution was mixed thoroughly and incubated. The mixture was transferred to an EPR flat cell for EPR data acquisition. 
<img src="/files/ftp_upload/2810/2810fig2.jpg" alt="Figure 2" /> 
Figure 2. EPR detection of NO from BAEC. (A) Spectrum of Fe2+-MGD only (B) Spectrum of Fe2+-MGD + CaI only. (C) Triplet spectrum resulting from NO trapping by Fe2+-MGD using CaI-stimulated cells. (D) Spectrum showing decrease in NO production due to 0.5 mM SIN-1 treatment of cells. 
<img src="/files/ftp_upload/2810/2810fig3.jpg" alt="Figure 3" /> 
Figure 3. EPR detection of DMPO-O2H from activated neutrophils. (A) Spectrum of 10 mM 
DMPO only. (B) Spectrum of PMN alone in the presence of 10 mM DMPO. (C) Spectrum of PMN activated by PMA in the presence of 10 mM DMPO.

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No conflicts of interest declared.

EPR spin trapping has been employed in a wide range of biomedical applications for quantifying and identifying free radicals. Spin trapping is highly sensitive, capable of detecting radicals at concentrations ranging from nM to &mu;M thus making it suitable for application in biological systems. The formation of the paramagnetic adduct, NO-Fe2+-MGD, is the basis of NO detection via EPR. Fe2+-MGD reacts with NO rapidly18 at a rate of ~ 106 M-1 s-1....

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td> Phenol free DMEM medium 
High glucose 1X</td>
 <td>GIBCO</td>
 <td>31053</td>
 <td></td>
 </tr>
 <tr>
 <td>0.25% Trypsin- EDTA</td>
 <td>GIBCO</td>
 <td>25200</td>
 <td></td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Fisher Scientific</td>
 <td>BP379-100</td>
 <td></td>
 </tr>
 <tr>
 <td>MEM Non Essential Amino acids</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovine serum</td>
 <td>Atlanta Biologicals</td>
 <td>S11550</td>
 <td></td>
 </tr>
 <tr>
 <td>Endothelial Growth factor</td>
 <td>Millipore</td>
 <td>02-102</td>
 <td></td>
 </tr>
 <tr>
 <td>CaI</td>
 <td>Enzo Life Sciences</td>
 <td>A-23187</td>
 <td>Dissolve in DMSO</td>
 </tr>
 <tr>
 <td>SIN-1</td>
 <td>Enzo Life Sciences</td>
 <td>BML-CN245-0020</td>
 <td></td>
 </tr>
 <tr>
 <td>DMPO</td>
 <td>Dojindo Laboratories</td>
 <td> D048-10 </td>
 <td></td>
 </tr>
 <tr>
 <td>FeSO4.7H2O</td>
 <td>Sigma Aldrich</td>
 <td>215422-250G</td>
 <td>Dissolve in PBS with Ca and Mg</td>
 </tr>
 <tr>
 <td>MGD</td>
 <td>Enzo Life Sciences</td>
 <td>ALX-400-014-M050</td>
 <td>Dissolve in PBS with Ca2+ and Mg2+</td>
 </tr>
 <tr>
 <td>BAEC cells</td>
 <td>Cell Systems</td>
 <td>2B2-C75</td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Fisher Scientific</td>
 <td>BP231-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Sigma Aldrich</td>
 <td>D8537</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS with CaCl2 and MgCl2</td>
 <td>Sigma Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Phorbol-myristate acetate (PMA)</td>
 <td>Sigma Aldrich</td>
 <td>79346-1MG</td>
 <td></td>
 </tr>
</tbody>
</table>

detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

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Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

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18.8K Views.  The Ohio State University. Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Video: Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...