RK and RRG were supported by a grant from the UNM Center for Metals in Biology and Medicine (CMBM) through NIH NIGMS grant P20 GM130422. RRG was supported by a pilot award from the NM-INSPIRES P30 grant 1P30ES032755. The imaging core support for the CX7 Cellomics instrument was provided through the AIM center cores funded by NIH grant P20GM121176.&#160;We would like to thank Dr. Sharina Desai and Dr. Li Chen for their invaluable assistance with technical issues related to the use of the CX7 Cellomics imaging platform.

1. Cell culture
<ol>
	<li>Seed the test cells (HepG2, HUH7, and JHH4 hepatocellular carcinoma cells) into a 96-well plate at a seeding density of 10,000 cells/well in a final seeding volume of 200 &#181;L per well.
	<ol>
		<li>Before culturing the HepG2 cells, coat the 96 plate wells with type IV collagen (50 &#181;g/mL) for 2 h duration at room temperature (RT). To avoid solidification of the stock collagen, place the stock solution into ice and subsequently, initiate the dilution process to the desired concentration.</li>
		<li>After a 2 h incubation period, aspirate off the excess collagen and wash the wells three times with the 1x PBS.</li>
	</ol>
	</li>
	<li>Cultivate the cells in Dulbecco&#39;s modified Eagle medium (DMEM) overnight at 37 &#176;C and 5% CO2 concentration in a humidified incubator.</li>
	<li>Next day, or upon reaching the 80%-90% confluency, whichever is earlier, treat the cells with H2O2&#160;(untreated, 250 &#181;M, 500 &#181;M, 750 &#181;M, and 1000 &#181;M) and Menadione (untreated, 25 &#181;M, 50 &#181;M, 75 &#181;M, 100 &#181;M), respectively for 30 min to induce the representative oxidative stress.</li>
	<li>Alternatively, choose to test the cells with the desired test substance of choice capable of inducing oxidative stress within the cells.</li>
</ol>
2. Stock and dilute solution preparation for DHE staining of cells
<ol>
	<li>Prepare DHE stock solution (5&#160;mg/mL or 15.9 mM) by dissolving 5 mg of DHE into 1 mL of DMSO.</li>
	<li>Dilute the DHE stock solution with double distilled autoclaved water to a final 100 &#181;M concentration.</li>
	<li>To avoid the freeze and thaw process of the dye (which may damage the fluorescence properties over time), prepare several aliquots in 1.5 mL centrifuge tubes with a concentration of 100 &#181;M and store them at -20 &#176;C.</li>
	<li>To achieve a final working concentration of 10 &#181;M, use an aliquot of the 100 &#181;M DHE concentration and dilute it with pre-warmed DMEM media. 
	NOTE: The working solution must be prepared fresh on the day of the experiment.</li>
	<li>Vortex the working fluorescence dye solution for 5-10 s to ensure proper mixing of the dye.</li>
</ol>
3. DHE staining process
<ol>
	<li>Remove the drug- or test-substance-containing media from each well and wash it gently once with either 1x PBS or DMEM. 
	NOTE: When performing the addition or washing steps in the experiment, it is crucial to avoid direct contact between the cells and the pipettor. This can be achieved by gently pipetting the PBS along the sides of the wells to minimize any potential mechanical damage to the cells. Ensuring that the pipettor does not directly touch the cells will help maintain the cell integrity and viability for the duration of the experiment.</li>
	<li>Add 100 &#181;L of DMEM media containing 10 &#181;M DHE (working solution) into each well and incubate at 37 &#176;C for 30 min.</li>
	<li>After 30 min incubation period, remove the DHE-containing media and wash each well gently three times with the 1x phosphate-buffered saline (PBS). This may be performed with a multi-channel pipettor to ensure rapid turnaround.</li>
	<li>Add 200 &#181;L of 1x PBS to each well with Hoechst 33342 nuclear staining dye for 10 min at RT.</li>
	<li>Remove the Hoechst 33342 nuclear staining solution from the well and add 200 &#181;L of 1x PBS to each well. 
	NOTE: To obtain fixed cells, follow the same protocol as for the live cells, with one additional step of adding 4% PFA for 5 min after treatment and DHE staining. Remove the PFA solution and wash cells with the 1x PBS. Then, incubate the cells with the nuclear staining dye for 10 min.</li>
	<li>Move the plate over to the high-content screening platform, fluorescence microscopy, or plate reader for image acquisition as quickly as possible.</li>
</ol>
4. Image acquisition and intensity measurement
<ol>
	<li>Plate loading
	<ol>
		<li>Open the Cellomics CX7 high-content screening (HCS) software for data acquisition.</li>
		<li>Carefully calibrate the imaging platform as per the manufacturer&#39;s specifications ahead of time with the specific 96-well plate of choice for use to avoid any hazy or blurry images in the data. 
		NOTE: Multi-well plates from various vendors may have different material properties and can influence the quality of the final images obtained.</li>
		<li>Once calibrated, save the system parameters specific to the plate brand in the instrument database and reuse them for future data acquisitions.</li>
		<li>Carefully place the plate with the prepared samples onto the heated stage of the HCS imaging system. Make sure that the plate is securely positioned and in the correct orientation for imaging on the microplate holder (i.e., locate the label marked A1 on the Reader stage, then rotate the microplate so that the location of well A1 matches the corner with the sticker).</li>
		<li>Gently press down on the microplate to ensure it rests flat against the stage. Uneven leveling of the plate in the HCS system can lead to errors in image acquisition.</li>
		<li>After the plate is in place, press and hold the Ctrl key, then click Ctrl-OK in the Plate Load/Unload dialog in the software.</li>
	</ol>
	</li>
	<li>Selecting imaging parameters
	<ol>
		<li>In the HCS imaging system, open Target Activation mode in the Cellomics bio-applications interface and create a new protocol using the two-channel setup protocol compatible with (a) nuclear staining and (b) DHE fluorescence probe data acquisition. In the current protocol, the filters&#160;386_BGSRS_BGSRS, 480_BGS_RS, and 386_BGS_RS&#160;were&#160;used for (a) Hoechst 33342 nuclear-staining, (b) total ROS production, and (c) superoxide radical detection, respectively. The choice of these filters was driven by the specific overlap for the emission properties of each dyes (DAPI and DHE) used as a part of this protocol. 
		NOTE: The naming convention of filters, e.g., 386-23_BGRFRN_BGRFRN, refers to the excitation of the sample with 386 nm light with a 23 nm bandwidth, followed by a dichroic filter that passes blue (B), green (G), red (R), far-red (FR), and near infra-red (N) light at specific wavelength ranges and the 386_BGS_RS refers to an emission filter that passes BGS and RS emission wavelength light after the dichroic.</li>
		<li>Specify the objectives for the image acquisition process within the software protocol interface, such as 10x or 20x magnification, as necessary.</li>
		<li>Choose the appropriate objective magnification depending on the desired level of imaging detail and resolutions necessary for the experiment. However, the resolution of each objective is fixed. Higher resolution detail will necessitate changing out the objective on the platform. In the current protocol, a 20x, 0.45 NA objective is used, which is a part of the high-content screening platform used in this protocol. 
		NOTE: The 20x objective represents a good trade-off between imaging resolution detail and the speed of acquisition necessary to capture ROS changes over time. Higher magnification objectives may be chosen (e.g., 40X), however, this will lead to increased acquisition times.</li>
		<li>Specify the exposure settings of the image acquisition camera, such as the exposure time, camera binning, and z-stack interval, according to the specific requirements of the protocol. 
		NOTE: The exposure time (often in milliseconds) varies based on the signal strength and sensitivity of the specific fluorophores used. This may also differ slightly from experiment to experiment based on the dye concentration and staining quality. In the current protocol, the parameters of acquisition are fixed as follows - Target % - 35%, image acquisition mode - 1104 x 1104 (with 2x2 binning), and objective 20x, 0.45 NA. These parameters may be set according to the specific experimental conditions.</li>
	</ol>
	</li>
	<li>Imaging configuration parameters 
	NOTE: Once the imaging parameters are set, the image collection process may be initiated using the software interface.
	<ol>
		<li>Image collection parameters
		<ol>
			<li>Identify the primary tracking object of interest (the nucleus of the cells) using different algorithms available in the instrument (optics - isodata, fixed, and triangle). 
			NOTE: The isodata method for the identification and marking of the primary object is used in this protocol.</li>
			<li>Segment the object (if any) either by shape or intensity parameter.</li>
			<li>Validate the primary object based on the desired size, shape, and intensity parameters unique to each cell type.</li>
			<li>Then, define the &#39;region of interest&#39; (ROI) around this primary object. 
			NOTE: The ROI is a key parameter of data acquisition that defines the location where the intensity of the fluorescence dye is identified to be consistent and repeatable across cell types. The ROI defines the key parameters (such as the intensity of the dye), which are measured for each primary tracking object (i.e., a cell) in different channels of the instrument. The ROI may be defined in the form of a circle or ring around the nucleus of each cell. The HCS software automatically obtains the intensity of the DHE fluorescence dye marker in channel 2 within the limit of the ROI for each cell that is segmented and analyzed in an automated manner.</li>
			<li>Set a 20 &#181;m circle around the primary object (nucleus). The distance of 20 &#181;m was chosen in this study based on the approximate sizes of the various cell lines used in this protocol (HepG2, JHH-4, and HUH-7). The 20 &#181;m ring ROI around the cells obtained the fluorescence intensity in a consistent and repeatable manner. 
			NOTE The key parameter in choosing an ROI is the ability to adequately cover the cell area; the size of the circular ROI depends on the cell size. Larger cells may require a larger ROI and vice-versa for a smaller ROI. These parameters must be determined ahead of time for each cell type and fluorescence dye of interest.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Population characterization and select features to store
	<ol>
		<li>Once the various acquisition parameters are defined in the HCS imaging protocol, set the HCS system into the data acquisition mode.</li>
		<li>The image acquisition parameters for this protocol were set as follows: a scan limit of 1000 cells/well, with a minimum requirement of 10 objects (cells) per field. 
		NOTE: The number of fields assessed for each well was determined based on the final cell count reaching the number 1000. The HCS system continues searching various locations within the well until it obtains the target population of 1000 cells/well. The acquisition speed thus depends upon the confluency and the total cell numbers present in each well of the 96-well plate. In the current protocol, the total- and average- intensity of channel 2 (representing the superoxide radicals and total ROS production) were collected from each well for further downstream analyses.</li>
	</ol>
	</li>
	<li>Image capture, processing, and analysis
	<ol>
		<li>After adjusting and finalizing the image acquisition parameters, initiate the scanning process for each 96-well plate. 
		NOTE: Cellomics CX7 imaging system enables high-content screening and automated analysis of samples of a variety of parameters, including ROS production within cells in a high-throughput manner. In addition to the ROS production, the HCS system is capable of providing additional valuable information about various parameters such as cell morphology, subcellular localization, and many other cellular features of interest. Once optimized for acquisition, the HCS imaging platform allows for comprehensive, qualitative, and quantitative characterization of cell parameters in a robust manner.</li>
		<li>After the scanning process is complete, launch the View Analysis software and export the various quantitative data parameters in a .csv format for additional analysis.</li>
		<li>Using third-party software, copy and paste the various quantitative values of interest for additional downstream analyses. 
		NOTE: In the current protocol, GraphPad Prism software was used to analyze the DHE intensity values in response to induced oxidative stress due to H2O2 and menadione exposures.</li>
	</ol>
	</li>
	<li>Data and statistical analysis
	<ol>
		<li>Calculate the mean average intensity of channel 2 (representing the total ROS values and superoxide radicals). 
		NOTE: All the experiments to calculate the intensity values were repeated three times with 6 replicates per condition at each dosing level.</li>
		<li>Perform one-way ANOVA or t-test to measure the differences in the intensity values between various test conditions.</li>
	</ol>
	</li>
</ol>

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

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The authors declare that they have no competing interests.

In this study, a protocol to assess superoxide-driven intracellular reactive oxygen species (ROS) production using dihydroethidium (DHE) fluorescence dye was established on a high-content screening system. A majority of the current protocols available in the literature use the DCFH-DA as a fluorescence imaging probe to quantify ROS species. However, multiple studies have shown the DCFH-DA is not an ideal probe for the measurement of intracellular ROS. Various reasons postulated for the unsuitability of DCFH-DA as a probe...

Reactive oxygen species (ROS) are a group of naturally occurring, highly reactive, and temporally unstable chemical radicals formed as a part of the normal cellular metabolism in&#160;cells. ROS plays a key and essential role in the modulation of normal physiological and biochemical processes occurring in cells1,2. The main source of ROS production in cells is from the mitochondrial electron transport chain (ETC) pathway as a part of the normal bioenergetic cycle. Significant additional sources of ROS production include enzymatic reactions such as cellular NADPH oxidases in cells. Metabolism of food molecules (e.g., glucose) occurs via the oxidative phosphorylation pathway in the mitochondrial matrix. A baseline level of ROS production is essential to regulate normal physiological cell signaling processes. Many key protein molecules that are part of the glucose metabolic signaling pathways (e.g., Akt and PTEN) are known to respond to intracellular ROS levels. Additionally, ROS are produced by various intracellular enzymes such as xanthine oxidase, nitric oxide synthase, and peroxisomal constituents as a part of the cellular enzymatic pathways1,2. In contrast to the natural sources of ROS, certain environmental factors, such as xenobiotics, infectious agents, UV light, pollution, cigarette smoking, and radiation, also lead to excessive production of ROS, which are a key driver of intra-cellular oxidative stress1,3. Elevated cellular oxidative stress can cause damage to native biomolecules inside a cell, such as lipids, proteins, and DNA, causing various diseases such as cancer, diabetes, cardiovascular disease, chronic inflammation, and neurodegenerative disorders1,3,4. Therefore, accurate measurements of ROS are essential to understand&#160;the cellular mechanisms involved in oxidative stress-induced disease pathophysiology.
Due to the short timescales of ROS production and elimination inside cells, quantitative measurements of various ROS radicals remains a challenge. Methods such as electron paramagnetic resonance (EPR)5, high-pressure liquid chromatography (HPLC), and fluorescence probe-based imaging are used to measure the various cellular ROS6. While methods such as EPR and HPLC yield quantitatively accurate estimates, these methods involve the destruction of the cellular spatial morphology and are usually in the form of global and bulk measurements of a sample. In contrast, imaging-based methods such as fluorescence probe-based methods retain the cellular morphology and spatial context of the ROS generation. However, the specificity of various fluorescence probes for different types of ROS radicals has not been well-established7,8. Several fluorescent probes such as dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (DCFH-DA), dihydrorhodamine (DHR), dimethyl anthracene (DMA), 2,7 dichlorodihydroflurescein (DCFH), 1,3-Diphenylisobenzofuran (DPBF), and MitoSox are available for ROS detection commercially. In the past decades, DHE, MitoSox, and DCFH-DA are the commonly used fluorescent dyes to measure ROS in cells and tissues8,9. DCFH-DA is a widely used dye for detecting intracellular H2O2 and oxidative stress. Despite the popularity of DCFH-DA, multiple previous studies have shown that it cannot be reliably used to measure intracellular H2O2 and other ROS levels8,9,10,11,12,13,14.
In contrast, the fluorescent probe dihydroethidium (DHE) shows a specific response to the intra-cellular superoxide radical (O2-). While the superoxide radical is one of many of the ROS species observed in cells, it is an important radical involved in the reduction of transition metals, conversion to peroxynitrate, and formation of hydroperoxides, among other intracellular effects. DHE is quickly taken up by the cells and has a fluorescence emission in the red wavelength range15. Upon reaction with superoxide radical specifically, DHE forms a red fluorescent product, 2-hydroxy ethidium (2-OH-E+). Thus, DHE may be considered as a specific probe for superoxide detection. However, DHE can also undergo nonspecific oxidation with ONOO- or OH., H2O2, and cytochrome c to form a second fluorescence product, ethidium E+, which can interfere with the measured 2-OH-E+ levels. However, these 2-OH E+ and E+ products, in combination, represent a major part of the total cellular ROS species observed inside a cell when stained with DHE. E+ intercalates into DNA, greatly enhancing its fluorescence8,9,10,11,13,14,15,16. Since the fluorescence spectra of ethidium and 2-hydroxy ethidium only differ slightly, the majority of ROS levels seen in a cell secondary to superoxide production can be detected and measured using DHE fluorescence products. These ROS species are identified using 480 nm wavelength excitation and 610 nm wavelength emission15,16,17.
In addition to choosing a specific fluorescent ROS detection probe, it is important to choose a sensitive method of detection to measure intracellular ROS. Accurate assessment of intracellular ROS levels is thus key to identifying disturbed redox balance states occurring in diseased cells or cells that have been exposed to various environmental stressors such as radiation, toxicological compounds, and genotoxic agents18. Since ROS is a commonly occurring phenomenon in cells that is responsible for regulating a variety of cell signaling activities, robust methods of detection of ROS are essential. To enable such high-throughput evaluation of ROS production within cells, this protocol uses a high-content screening (HCS) platform to measure the ROS species. The current protocol allows the high-throughput analysis of intracellular ROS production, which is of critical importance in many toxicology studies19. This protocol aims to provide an easy and versatile solution to detect and measure intracellular ROS in adherent hepatocellular carcinoma cells. The chemical reagents of H2O2 and menadione are used as potent ROS stimulators to measure the relative levels of ROS production in a controlled and high throughput setting. This protocol may be fine-tuned to measure ROS production in adherent and nonadherent cells under appropriate conditions, as necessary.

<table><tbody><tr><td>1.5 mL centrifuge tubes&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>20170-038&amp;nbsp;</td><td></td></tr><tr><td>96- well plate&amp;nbsp;</td><td>Corning Costar&amp;nbsp;</td><td>07-200-90&amp;nbsp;</td><td></td></tr><tr><td>Cellomics Cx7</td><td>ThermoFisher&amp;nbsp;</td><td>HCSDCX7LEDPRO</td><td></td></tr><tr><td>Collagen&amp;nbsp;</td><td>Advanced Biomatrix&amp;nbsp;&amp;nbsp;</td><td>5056&amp;nbsp;</td><td></td></tr><tr><td>DHE (Dihydroethidium)&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>D1168&amp;nbsp;</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Sigma&amp;nbsp;&amp;nbsp;</td><td>6046&amp;nbsp;</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>97068-085&amp;nbsp;</td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>Version 6.0</td><td></td></tr><tr><td>HepG2 cell line</td><td>ATCC</td><td></td><td></td></tr><tr><td>Hoechst&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>33342&amp;nbsp;</td><td></td></tr><tr><td>HUH7 cell line</td><td>ATCC</td><td></td><td></td></tr><tr><td>Hydrogen Peroxide&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>88597&amp;nbsp;</td><td></td></tr><tr><td>JHH4 cell line</td><td>ATCC</td><td></td><td></td></tr><tr><td>Menadione&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>M5625&amp;nbsp;</td><td></td></tr></tbody></table>

high throughput screening assessment of reactive oxygen species (ros) generation using dihydroethidium (dhe) fluorescence dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

Dihydroethidium (DHE) is a superoxide-responsive fluorescence dye that provides specific information regarding the intracellular ROS states. DHE dye intrinsically emits blue fluorescence in the cytoplasm. However, upon interaction with superoxide radicals, it is transformed into 2-hydroxyethidium, which emits fluorescence in the red wavelengths (&#62;550 nm) (Figure 1). DHE dye is easily transported into the cells and the nucleus. The fluorescence emitted can be visualized with a fluorescenc...

Watch this Scientific Journal Video about High Throughput Screening Assessment of Reactive Oxygen Species ROS Generation using Dihydroethidium DHE Fluorescence Dye at JoVE.com

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

high-throughput-screening-assessment-reactive-oxygen-species-ros

Research

JoVE Journal

Bioengineering

4.0K Views.  University of New Mexico. This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Video: Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Cancer Research

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Biochemistry

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

Neuroscience

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Biology

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are many deleterious effects associated with unchecked ROS production, including structural damage to DNA and other biological molecules. However, ROS can also be mediators of physiological signaling. There is accumulating evidence that ROS play significant roles in everything from activation of transcription factors to the mediation of inflammatory toxicity that kills foreign pathogens and defend the body.In this video we will delve into the associations between ROS, metabolism and disease. After establishing their significance, we will discuss the principles and a protocol of a commonly used methodology for measuring ROS levels in cells: the use of non-fluorescent probes that become fluorescent upon oxidation. Lastly, we will review some current applications of this technique in cell biology research.

Reactive Oxygen Species and Oxidative Stress

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...