The authors acknowledge the support from the National Natural Science Foundation of China (32070768, 31871404, 31900658, 32270754) and the State Key Laboratory of Drug Research.

The Animal Care Committee of Tongji University approved the present work, and all animal care guidelines were followed. Male or female C57BL/6J mice between 8-12 weeks of age were used for the experiments. It was ensured that the age and sex were the same in the experimental groups; otherwise, the susceptibility to the disease was affected. Mice were housed in a specific pathogen-free environment with alternating 12 h light and dark cycles under constant conditions (room temperature 23 &#177; 1 &#176;C, humidity 50% &#177; 10%), with free access to mouse food and water.
1. Preparation of MOG35-55 emulsion
<ol>
	<li>Add heat-inactivated lyophilized Mycobacterium tuberculosis (MTB, H37Ra) to complete Freund&#39;s adjuvant (itself containing 1 mg/mL of heat-inactivated MTB, H37Ra), resulting in a final MTB concentration of 5 mg/mL (see Table of Materials). 
	NOTE: The entire operation must be completed in the biosafety cabinet; do not open the blowing air.</li>
	<li>Dissolve the lyophilized MOG35-55 peptide (see Table of Materials) with sterile pre-cooled Phosphate Buffered Saline (PBS) (without calcium and magnesium ions, pH 7.4) to prepare the antigen solution at the concentration of 2 mg/mL.</li>
	<li>Take a clean 2 mL microcentrifuge tube and add one sterilized 5 mm steel ball (see Table of Materials) to each tube.</li>
	<li>Add 500 &#181;L of complete Freund&#39;s adjuvant containing 5 mg/mL of MTB and 500 &#181;L of MOG35-55 antigen solution to the above microcentrifuge tube containing one steel ball.</li>
	<li>Oscillate the above tube on a TissueLyser (see Table of Materials) for 10 min, cool on ice for 10 min, and repeat four times to mix it well and finally form a white viscous solution. 
	NOTE: Good emulsification is a key step in preparing MOG35-55 emulsion, so thorough mixing is required. The TissueLyser is set to a speed of 28 Hz.</li>
</ol>
2. Preparation of pertussis toxin (PTX)
<ol>
	<li>Prepare PTX with ddH2O into a 100 &#181;g/mL concentration and store at 4 &#176;C.</li>
	<li>Dilute the PTX stock solution 50 times with sterile 1x PBS (without calcium and magnesium ions, pH 7.4) to make a 200 ng/100 &#181;L solution for use.</li>
</ol>
3. Establishment of EAE animal model
<ol>
	<li>Construct the EAE model using the 8-12-week-old male or female C57BL/6J mice. Ensure that the mice are adequately acclimatized to the feeding environment prior to immunization.</li>
	<li>Centrifuge the prepared MOG35-55 emulsion (step 1) at 4 &#176;C for 2-3 s by pressing the Pulse button of the equipment (see Table of Materials) to precipitate all the emulsions at the bottom of the tube. 
	NOTE: MOG35-55 emulsion can be stored at -20 &#176;C for several days. To avoid drug failure, it is recommended to use it as soon as possible.</li>
	<li>Attach a 22 G needle to a 1 mL syringe barrel, aspirate the MOG35-55 emulsion, and transfer the MOG35-55 emulsion into a new 1 mL syringe barrel. Secure the connection between the 1 mL syringe barrel and a 26 G needle with sealing film (see Table of Materials). 
	NOTE: Avoid air bubbles when loading MOG35-55 emulsion into 1 mL syringe barrels.</li>
	<li>Wipe and disinfect the&#160;injection site with 70% ethanol.</li>
	<li>Inject the MOG35-55 emulsion subcutaneously on each side of the dorsal spine of the mice, 100 &#181;L on each side. Observe the automatic formation of bulbous masses under the skin of the mice&#39;s dorsum after the injection operation is completed. 
	NOTE: Ensure that experienced experimenters perform the immunization process and that the injection is done gently and slowly to minimize the pressure on mice.</li>
	<li>Inject the above mice intraperitoneally with 100 &#181;L of PTX (step 2). 
	NOTE: The day of immunization is day 0. Also, ensure that the mice can be accurately identified for subsequent daily evaluation, such as using a color marker on the tail of the mice.</li>
	<li>Inject the same dose of PTX on day 2 after immunization.</li>
	<li>Prepare a group of unimmunized mice as wild-type (WT) mice.</li>
</ol>
4. Clinical monitoring of mice
<ol>
	<li>Record the bodyweight of EAE and WT mice daily. 
	NOTE: The severity of EAE is positively correlated with the weight loss of mice, so bodyweight is also a very important monitoring index.</li>
	<li>Monitor the status of mice from 0-21 days after immunization using the 0-5 scoring system listed in Table 1. 
	NOTE: Symptoms in between are counted as plus or minus 0.5 points.</li>
</ol>
5. Open field test
NOTE: The experimental animals selected for this step are EAE mice in the early onset, peak, and remission periods. In addition, WT mice were used as control. It is to be noted that all mice were tested for anxiety-like behavior prior to modeling to exclude mice with anxiety disorders for EAE modeling. In addition, EAE mice in peak and remission periods with complete motor incapacity were excluded from the test.
<ol>
	<li>Prepare a 40 &#215; 40 &#215; 40 cm3 open field reaction chamber and a locomotion activity (open field) video analysis system (see Table of Materials). 
	NOTE: The camera is installed in a position that completely covers the box, the reaction room is evenly lit, and the test room is required to be a quiet area.</li>
	<li>Place the test mice in the test room for habituation 1 h before starting the experiment.</li>
	<li>Spray the entire area with 70% ethanol and wipe with a clean paper towel to ensure the reaction chamber is clean before starting the test.</li>
	<li>Remove each mouse individually from its cage and place it in the same corner of the arena before starting to explore. 
	NOTE: The bottom of the box is divided into 16 grids, of which the middle four grids area is the central area and the surrounding area is the peripheral area.</li>
	<li>Click on the Start Capture button in the menu bar of the video analysis system, record time, and begin shooting.</li>
	<li>Keep quiet in the test room.</li>
	<li>Let the mouse move freely for 5 min during the recording process.</li>
	<li>Stop the acquisition system and save the video.</li>
	<li>Take the mouse out of the arena, put it back in the cage, and proceed to the next mouse. 
	NOTE: Clean the test area with 70% ethanol between runs to remove odors and other substances.</li>
	<li>Analyze the results using the video analysis system.</li>
</ol>
6. Analysis of bone phenotype
<ol>
	<li>Euthanize the EAE and WT mice by cervical dislocation on the 21st day. 
	NOTE: Personnel performing cervical dislocation operations must be well trained to minimize the pain endured during the animal&#39;s death.</li>
	<li>Make the mouse lie flat in a dissecting tray and fix the extremities.</li>
	<li>Hold the mouse hind limb skin with forceps and open the mouse skin and muscle tissue with scissors.</li>
	<li>Separate the femur from the tibia and hip bone carefully with scissors.</li>
	<li>Remove the muscle adhering to the femur with scissors and place the femur in 70% ethanol at room temperature.</li>
	<li>Scan the distal femur using a micro-CT system (see Table of Materials) with an isotropic voxel size of 10 &#181;m, with a peak X-ray tube voltage of 70 kV and an X-ray intensity of 0.114 mA. 
	NOTE: A 3D Gaussian filter allows the denoising of 2D threshold images.</li>
	<li>Analyze the 100 slices scanned from the middle femoral shaft to measure femur parameters, including bone volume, tissue volume, bone mineral density, trabecular separation, trabecular number, trabecular connection density, and trabecular and cortical thickness. 
	NOTE: Starting from the proximal end of the distal femur growth plate in mice, sections completely devoid of epiphyseal cap structures were found and continued to extend 100 slices towards the proximal femur, which were manually outlined contours at several voxels away from the inner cortical surface to identify epiphyseal trabeculae.</li>
	<li>Create the 3D reconstructions by stacking threshold 2D images from contour regions in the micro-CT system.</li>
</ol>

The authors have nothing to disclose.

MS is a demyelinating inflammatory disease of the CNS and is one of the most common neurological disorders causing chronic disability in young people, imposing a huge burden on families and society3,4. MS has always been classified as an organ-specific T cell-mediated autoimmune disease, inducing the autoimmune system to slowly erode CNS, which will involve multiple systems throughout the body27. Typical clinical symptoms include visual im...

Autoimmune diseases are a spectrum of disorders caused by the immune system&#39;s immune response to its own antigens resulting in tissue damage or dysfunction1. Multiple sclerosis (MS) is a chronic autoimmune disease of polyneuropathy in the central nervous system (CNS), characterized by inflammatory infiltration, demyelination, and neuronal axonal degeneration2,3. At present, MS has affected as many as 2.5 million people around the world, mostly young and middle-aged people aged 20-40, who are often the backbone of their families and society. This has caused considerable impact and harm to families and society2,4.
MS is a multifactorial disease with diverse and complex clinical manifestations. In addition to classic neurological disorders characterized by inflammatory infiltration and demyelination, MS often shows visual impairment, limb dyskinesia, and cognitive and emotional disorders5,6,7. If MS patients do not get the proper and correct treatment, half of them will live in wheelchairs after 20 years, and nearly half of them will experience depressive and anxiety symptoms, leading to much higher levels of suicidal ideation than the general population8,9.
Despite a long research period, the etiology of MS remains elusive, and the pathogenesis of MS has not yet been elucidated. Animal models of MS have allowed serving as testing tools to explore disease development and new therapeutic approaches, despite the significant differences between the rodent and human immune systems, while at the same time sharing some basic principles. Experimental autoimmune encephalomyelitis (EAE) is currently the ideal animal model for studying MS, which uses autoantigen immunity from myelin proteins to induce autoimmunity to CNS components in susceptible mice, with the addition of complete Freund&#39;s adjuvant (CFA) and pertussis toxin (PTX) to enhance the humoral immune response. Depending on the genetic background and immune antigens, different disease processes, including acute, relapsing-remitting, or chronic, are obtained to mimic various clinical forms of MS10,11,12. The relevant immunogens commonly used in the construction of EAE models come from self-CNS proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG). MBP- or PLP-immunized SJL/L mice develop a relapsing-remitting course, and MOG triggers chronic progressive EAE in C57BL/6 mice11,12,13.
The main purpose of disease-modifying therapy (DMT) is to minimize disease symptoms and improve function6. Several drugs are used clinically to alleviate MS, but no drug has yet been used to completely cure it, revealing the necessity of synergistic treatment. C57BL/6 mice are currently the most commonly used to construct transgenic mice, and in this work, an EAE model induced by MOG35-55 in C57BL/6J mice with a 5-point scale was used to monitor the disease progression. EAE models also suffer from anxiety-like moods and bone loss, and the widely known demyelinating lesions. Here, the method to assess the symptoms of EAE from multiple perspectives using open-field test and micro-computed tomography (Micro-CT) analysis is also described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe(with 26 G needle)</td>
			<td>Shanghai Kindly Medical Instruments Co., Ltd</td>
			<td>60017031</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tube</td>
			<td>HAIKELASI</td>
			<td>KY-LXG2A</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G needle</td>
			<td>Shanghai Kindly Medical Instruments Co., Ltd</td>
			<td>60017208</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Freund&#8217;s Adjuvant</td>
			<td>Sigma</td>
			<td>F5881</td>
			<td>Stored at 4 &#176;C, 1 mg of heat-inactivated MTB (H37Ra) per mL</td>
		</tr>
		<tr>
			<td>Conditioned place preference system</td>
			<td>Shanghai Jiliang Software Technology Co., Ltd</td>
			<td></td>
			<td>Animal behavior</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10009218</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Locomotion activity (open field) video analysis system</td>
			<td>Shanghai Jiliang Software Technology Co., Ltd</td>
			<td>DigBehv-002</td>
			<td>Animal behavior</td>
		</tr>
		<tr>
			<td>MOG35-55 peptide</td>
			<td>Gill Biochemical Co., Ltd</td>
			<td>GLS-Y-M-03590</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37Ra</td>
			<td>BD</td>
			<td>231141</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Open field reaction chamber</td>
			<td>Shanghai Jiliang Software Technology Co., Ltd</td>
			<td></td>
			<td>Animal behavior</td>
		</tr>
		<tr>
			<td>Pertussis toxin</td>
			<td>Calbiochem</td>
			<td>516560</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td></td>
			<td></td>
			<td>Made in our laboratory</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Shanghai Medical Instrument (group) Co., Ltd</td>
			<td>J21010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing film</td>
			<td>Heathrow Scientific</td>
			<td>HS 234526B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21R Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002447</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel ball</td>
			<td>QIAGEN</td>
			<td>69975</td>
			<td></td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>QIAGEN</td>
			<td>85300</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Shanghai Medical Instrument (group) Co., Ltd</td>
			<td>JD1060</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;CT 35 desktop microCT scanner</td>
			<td>Scanco Medical AG, Bassersdorf, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and diverse assessment indicators of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

After immunization of the mice, the bodyweight of the mice is recorded daily, and their clinical symptoms are evaluated according to the protocol described above (step 4). In C57BL/6J mice immunized with MOG peptide, because the location of the lesion is mainly confined to the spinal cord,the pathogenesis of EAE mice spreads from the tail end to the head. At the beginning of the disease, EAE mice exhibit weakness and drooping of the tail, followed by weakness of the hind limbs, uncoordinated movement, and paralysis. As t...

Watch this Scientific Journal Video about Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis at JoVE.com

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

induction-diverse-assessment-indicators-experimental-autoimmune

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3.2K Views.  Tongji University. The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Video: Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence).
Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs.
The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.

This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by ...

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Experimental Approaches to Tissue Engineering

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...