We are grateful to Drs Karl-Heinz Krause and Vincent Jaquet for help and advice to 
set up these protocols, and also thank Christoph Bauer and J&eacute;r&ocirc;me Bosset from the Bioimaging Platform of the NCCR and Dr. Navin Gopaldass for their technical support. The research is supported by a ProDoc grant of the Swiss National Science Foundation.

1. Visualization and Qualitative Measurement of ROS Production in Phagosomes
The OBG-coated beads should be prepared in advance. The beads coating procedures and agar overlay technique are adapted from published references5,14.
<ol>
	<li>Add 1 ml suspension of 3.0 &#956;m carboxylated silica beads (about 1.8 x 109 beads) into an 1.5 ml tube, wash the beads 3x with 1 ml of PBS by quick spin and vortexing.</li>
	<li>Resuspend the beads in 1 ml of PBS containing 25 mg/ml cyanamide (to activate the carboxylated silica beads to covalently bind prelabeled BSA), incubate on a wheel for 15 min.</li>
	<li>Wash 3x with 1 ml of coupling buffer (0.1 M sodium borate, pH 8.0) by quick spin (centrifuge at full speed in a table top mini centrifuge for 5 sec or alternatively centrifuge at 2,000 x g for 1 min in a table top centrifuge) and vortexing to remove excess cyanamide.</li>
	<li>Mix the washed beads with 500 &#956;l of coupling buffer containing 1 mg of OxyBurst Green (OBG) H2HFF (dihydro-2&#39;,4,5,6,7,7&#39;-hexafluorofluorescein)-BSA, fill the tube with nitrogen gas from a standard gas-can before capping, and incubate on a wheel for 14 hr (overnight) at RT in the dark.</li>
	<li>Wash the beads 2x with 1 ml of quenching buffer (250 mM glycin in PBS) to remove unreacted OBG, and twice with coupling buffer to remove the quenching buffer&#160;by quick spin and vortexing.</li>
	<li>Add 1 ml of coupling buffer containing 50 &#956;g of Alexa fluor 594 succinimidyl ester to conjugate to BSA. Fill the tube with nitrogen before capping, and incubate on a wheel for 1.5 hr at room temperature in the dark.</li>
	<li>Wash the beads 3x with 1 ml of quenching buffer by quick spin and vortexing to stop the reaction, then wash the beads 3x with 1 ml of PBS by quick spin and vortexing.</li>
	<li>Finally, resuspend the beads in 1 ml of PBS with 2 &#956;l of 10% w/v azide for long-term storage. Measure the concentration of beads in the suspension with a hemocytometer (usually around 1-2 x 109 beads/ml), fill the tube with nitrogen before capping, and store at 4 &#176;C in the dark.</li>
	<li>The coating efficiency of the OBG fluorescein can be tested by checking the emission spectrum using excitation at 500 nm. When oxidized by H2O2 in the presence of horseradish peroxidase (HRP), the fluorescence intensity of oxidized beads, at emission peak (538 nm), is 11-12x&#160;higher than that of nonoxidized coated beads.</li>
	<li>Harvest exponentially growing Dictyostelium cells from a 10 cm Petri dish, plate different densities of cells on 3 cm dishes with an optically clear plastic or glass bottom, and grow them overnight. Choose the dishes that are about 80% confluent for the experiment. A detailed protocol for cultivating Dictyostelium cells has been published15.</li>
	<li>Replace the culture medium with LoFlo medium (LF medium), incubate for 2 hr before the experiment in order to decrease the extracellular and intraendosomal autofluorescence of the HL5C culture medium.</li>
	<li>In parallel, melt 10 ml 1.5% bacto agar in LF medium, pour the agar onto a flat surface (i.e. a glass plate of 10 cm x 10 cm) in order to form an agar layer about 1 mm thick, wait for 10-15 min to solidify. Cut the agar layer into 2 cm x 2 cm squares and place in LF medium for later use.</li>
	<li>Meanwhile, prepare the spinning-disk or wide field microscope, set the temperature of the environmental chamber at 22 &#176;C and adjust the settings for this experiment. (See additional comments in the discussion).</li>
	<li>After 2 hr of incubation, aspirate the LF medium from the 3 cm dish, but the cell monolayer should still be covered by a thin film of medium. Dilute the coated beads to 1.5 x 107 beads/ml, and add 10 &#956;l onto the cell layer.</li>
	<li>Take one square agar sheet, drain excess liquid but keep wet. Gently put the agar square on top of the cell layer. Do not move the agar square when it is lying on the cells. The agar overlay is used to increase contact between beads and cells, thereby improving uptake, and it also slightly compresses the cells, keeping them better in the focal plane of the objective.</li>
	<li>Place the lid onto the dish, place it on the microscope stage, and automatically take pictures in the red, green and phase channels every 1 min for 2 hr or longer.</li>
	<li>Select and focus on cellular events that contain the whole process of phagocytosis. Merge the optimized 3 channels and assemble the pictures into a movie using professional image processing software. Quantify fluorescence intensities of each selected beads in red and green channels, and the ratio of green/red will reflect the dynamic phagosomal ROS production of the cells.</li>
</ol>
2. Quantitative and Medium-throughput Measurement of Intracellular Superoxide Production
<ol>
	<li>Collect one 80% confluent dish of Dictyostelium in 10 ml of HL5C medium. Centrifuge Dictyostelium cells at 850 x g for 5 min, carefully and completely aspirate excess medium. Resuspend cells in SS6.4 buffer [0.12 M sorbitol in Sorensen buffer (14.69 mM KH2PO4 and 6.27 mM Na2HPO4), pH 6.4], count cells and dilute to a final density of 6 x 106 cells/ml (see additional comments in the discussion).</li>
	<li>Add 50 &#956;l of cell suspension into each well of a white nontransparent 96 well plate (see additional comments in the discussion).</li>
	<li>Dilute DHE stock (30 mM in DMSO) 500 fold with SS6.4, pipette 50 &#956;l of diluted DHE into each well using a multichannel pipette if necessary. The final concentration of DHE is 30 &#956;M. The reaction starts immediately.</li>
	<li>Stimuli or inhibitors can be added at this point, or at other time points according to specific needs.</li>
	<li>Use the end point &#34;top reading&#34; mode of a fluorescence microplate reader, with fluorescence excitation/emission at 522 nm/605 nm, read every 2 min for 1 hr, medium shake 5 sec/min, at 22 &#176;C.</li>
</ol>
3. Quantitative and High Throughput Measurement of Extracellular H2O2 Production
<ol>
	<li>Follow the same steps as described in 2.1 and 2.2.</li>
	<li>Prepare diluted HRP by adding 5 &#956;l of HRP stock (100 U/ml in distilled water) into 10 ml of SS6.4, vortex or invert the tube to mix well and keep on ice for later use. The diluted HRP solution is at 0.05 U/ml.</li>
	<li>Prepare the AUR reaction mixture by mixing the AUR stock (10 mM AUR in DMSO) and the diluted HRP solution into SS6.4 buffer to the final concentrations of 6.25 &#956;M, and 0.005 U/ml, respectively. As an example, to prepare the reaction mixture for 40 reactions, mix 1600 &#956;l of SS6.4 with 400 &#956;l of diluted HRP and 2.5 &#956;l of AUR stock solution.</li>
	<li>Add 50 &#956;l of AUR mixture into each well using a multichannel pipette if necessary. Now the reaction starts.</li>
	<li>Stimuli or inhibitors can be added at this point, or at other time points according to specific needs.</li>
	<li>Use the end point &#34;top reading&#34; mode of a fluorescence micro plate reader, with fluorescence excitation/emission at 530 nm/590 nm, read every 2 min for 1 hr, medium shake 5 sec/min, at 22 &#176;C.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+superoxide+and+hydrogen+peroxide+detection+in+biological+assays.">Enhanced superoxide and hydrogen peroxide detection in biological assays.</a> Free Radic. Biol. Med. 49, 61-66 (2010).</li><li>Chen, J., Rogers, S. C., Kavdia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Kinetics+of+Dihydroethidium+Fluorescence+with+Superoxide+Using+Xanthine+Oxidase+and+Hypoxanthine+Assay.">Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay.</a> Ann. Biomed. Eng. , (2012).</li><li>Lee, C. W., Chen, Y. C., Ostafin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+accuracy+of+Amplex+Red+assay+for+hydrogen+peroxide+in+the+presence+of+nanoparticles.">The accuracy of Amplex Red assay for hydrogen peroxide in the presence of nanoparticles.</a> J. Biomed. Nanotechnol. 5, 477-485 (2009).</li><li>Guimaraes-Ferreira, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+creatine+supplementation+decreases+reactive+oxygen+species+content+with+no+changes+in+expression+and+activity+of+antioxidant+enzymes+in+skeletal+muscle.">Short-term creatine supplementation decreases reactive oxygen species content with no changes in expression and activity of antioxidant enzymes in skeletal muscle.</a> Eur. J. Appl. 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No conflicts of interest declared.

Compared to the previously described methods, the OBG assay dynamically visualizes the process of phagosomal ROS generation at the single cell level, instead of measuring an average ROS signal from a population of cells. Such population-averaging methods tend to obscure critical information caused by nonsynchronous phagocytosis. We successfully adapted DHE and AUR assays to Dictyostelium and optimized the protocols. Most importantly, we specified the types and subcellular localizations of ROS measured by these t...

Reactive oxygen species (ROS) are involved in a wide variety of biological processes such as host defense, signaling, tissue development and response to injury as well as hypertension and cancer. The well-studied phagosomal NADPH oxidase machinery is dedicated to rapid ROS generation, known as the oxidative burst, to kill bacteria ingested in neutrophils&#39; phagosomes1. In addition, leakage of electrons as a byproduct of the mitochondrial respiratory chain was previously thought to be responsible only for an unregulated source of ROS. But recently, it was identified as an important mechanism to contribute to intraphagosomal killing of bacteria in mouse macrophages2. In recent years, the social amoeba, Dictyostelium, has become a powerful and popular model to study cell intrinsic mechanisms of the innate immune response. Indeed, Dictyostelium and human phagocytes share a surprisingly high level of conservation in molecular machineries responsible for bacteria sensing, engulfment and killing3,4. The homologs of proteins and enzymes related to ROS production or detoxification, such as NADPH oxidases, catalases, superoxide dismutases, can be found in both human and Dictyostelium. As the best studied amoeba, Dictyostelium presents several unique advantages over mammalian model system. They grow at room temperature without the need for CO2, with a doubling time of 8-10 hr. They can be easily kept as adherent or suspension cultures. In addition, thanks to their fully sequenced and annotated haploid genome, and to easy genetic manipulation, Dictyostelium has become a very attractive experimental model organism.
In previous studies, various chlorinated and fluorinated derivatives of fluorescein (collectively known as OxyBurst Green, OBG) that emit fluorescence after oxidation by ROS, have been used as a ROS reporter. Cell-permeant esterified derivatives are used to measure cytoplasmic ROS, whereas cell-impermeant dextran- or protein-coupled derivatives are used to measure extracellular ROS. In particular, OBG BSA-coated beads have already been used to detect phagosomal ROS production in mammalian cells5. However, the microplate reader-based approach can only give an averaged ROS generation curve from a population of cells. With the present protocol, by using Dictyostelium , a professional phagocyte, and optimized experimental conditions, we obtain stable and efficient phagocytosis without conjugating any opsonin onto the beads. DHE has been used in various model systems, such as mammalian neutrophils and macrophages, to detect ROS production6-9. Meanwhile, there are some controversies over the specificity and sensitivity of the method8,10. As an improved version of Amplex Red, the fluorescence intensity of AUR is less sensitive to pH, which makes it more suitable to measure ROS in nonneutral or weakly acidic milieus. AUR has been recently applied in several mammalian systems11,12, but its use in nonmammalian models, which quite often require weakly acidic growth media, has not been reported yet. In addition, there is no published protocol to quantitatively and dynamically measure ROS production and localization in the social amoeba Dictyostelium.
The goal of the presented set of protocols is to provide an easy and versatile solution to monitor various ROS and their localization, and further give insights into ROS-related cellular mechanisms. For the OBG assay, we used live microscopy to monitor the whole process of phagosomal ROS generation after uptake of OBG-coated beads by Dictyostelium cells, and provided a new approach to study the mechanism of intraphagosomal killing of bacteria. We have optimized medium-throughput DHE and AUR assays in Dictyostelium, to measure intracellular superoxide and extracellular H2O2 production, respectively, by using LPS as a potent ROS stimulator. Note that LPS was recently shown to increase the bactericidal activity of Dictyostelium towards phagocytosed bacteria13. In addition, treatment with DEDTC and catalase explicitly confirmed that these two methods specifically measure different types and subcellular localizations of ROS in Dictyostelium. Finally, the OBG assay can be adapted to any adherent phagocytic cell, by further opsonizing the beads with ligands for phagocytic receptors of animal cells. In principle, the DHE and AUR assays can also be fine-tuned for measurements in any adherent or nonadherent cell under appropriate experimental conditions.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>OxyBURST Green H2HFF BSA</td>
			<td>Invitrogen</td>
			<td>O-13291</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-20004</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylated silica beads</td>
			<td>Kisker Biotech</td>
			<td>PSi-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>HL5C medium</td>
			<td>ForMedium</td>
			<td>HLC0102</td>
			<td></td>
		</tr>
		<tr>
			<td>LoFlo medium</td>
			<td>ForMedium</td>
			<td>LF1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>204010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium</td>
			<td>Sigma</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed</td>
			<td>Invitrogen</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Roche</td>
			<td>10108090001</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyldithiocarbamate (DEDTC)</td>
			<td>Sigma</td>
			<td>D3506-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C9322-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanamide</td>
			<td>Sigma</td>
			<td>187364-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L2630-25G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>ibidi &#956;-Dish 35 mm, high</td>
			<td>ibidi</td>
			<td>81156</td>
			<td>Bottom made of optically clear plastic</td>
		</tr>
		<tr>
			<td>MatTek dish 35 mm</td>
			<td>MatTek corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Bottom made of a glass coverslip</td>
		</tr>
		<tr>
			<td>Scepter Cell Counter</td>
			<td>Merck Millipore</td>
			<td>PHCC00000</td>
			<td></td>
		</tr>
		<tr>
			<td>White 96 well plate</td>
			<td>Nunc</td>
			<td>236108</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SORVALL</td>
			<td>Legend RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy Mx</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The generation of ROS in phagosomes can be visualized qualitatively and dynamically by microscopy (File S1). The red fluorescence emitted by Alexa fluor 594 is pH-insensitive and remains constant in the phagosomal environment, while oxidation of OBG increases its fluorescence in the green channel. The emission spectra of in vitro oxidized and nonoxidized OBG-coated beads are compared in Figure 1B, showing a significant increase in intensity after oxidation. To facilitate visuali...

Watch this Scientific Journal Video about Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System at JoVE.com

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

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 ...

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16.0K Views.  University of Geneva. 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.

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

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

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 ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...