The authors thank Lab Animal Resources for their animal husbandry. Our funding sources for this manuscript include the Boettcher Fund, the Murphy Turner Fund, CSU College of Veterinary Medicine, and the Biomedical Sciences College Research Council. Figure 2A, Figure 2C, and Figure 3A were created with BioRender.com.

Mice were bred and maintained at Colorado State&#39;s Lab Animal Resources, accredited by the Association for Assessment and Accreditation of Lab Animal Care International, in accordance with protocol #1138, approved by the Institutional Animal Care and Use Committee at Colorado State University.
1. Isolating and infecting primary cortical mixed glia with prions
<ol>
	<li>To isolate primary mixed glia containing both astrocytes and microglia, obtain C57Bl/6 mouse pups aged z...

Coculturing AdMSCs and Glia to Reduce Prion-Induced Inflammation

<ol>
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A., Race, B., Williams, K., Striebel, J., Chesebro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+are+critical+in+host+defense+against+prion+disease.">Microglia are critical in host defense against prion disease.</a> Journal of Virology. 92 (15), e00549 (2018).</li><li>Bradford, B. M., McGuire, L. I., Hume, D. A., Pridans, C., Mabbott, N. 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D., 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=Intranasally+delivered+mesenchymal+stromal+cells+decrease+glial+inflammation+early+in+prion+disease.">Intranasally delivered mesenchymal stromal cells decrease glial inflammation early in prion disease.</a> Frontiers in Neuroscience. 17, 1158408 (2023).</li><li>English, K., Barry, F. P., Field-Corbett, C. P., Mahon, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma+and+TNF-alpha+differentially+regulate+immunomodulation+by+murine+mesenchymal+stem+cells.">IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells.</a> Immunology Letters. 110 (2), 91-100 (2007).</li><li>Hemeda, H., 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=Interferon-gamma+and+tumor+necrosis+factor-alpha+differentially+affect+cytokine+expression+and+migration+properties+of+mesenchymal+stem+cells.">Interferon-gamma and tumor necrosis factor-alpha differentially affect cytokine expression and migration properties of mesenchymal stem cells.</a> Stem Cells and Development. 19 (5), 693-706 (2010).</li><li>Carta, M., Aguzzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+foundations+of+prion+strain+diversity.">Molecular foundations of prion strain diversity.</a> Current Opinion in Neurobiology. 72, 22-31 (2022).</li><li>Yu, F., 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=Phagocytic+microglia+and+macrophages+in+brain+injury+and+repair.">Phagocytic microglia and macrophages in brain injury and repair.</a> CNS Neuroscience and Therapeutics. 28 (9), 1279-1293 (2022).</li><li>Sinha, A., 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=Phagocytic+activities+of+reactive+microglia+and+astrocytes+associated+with+prion+diseases+are+dysregulated+in+opposite+directions.">Phagocytic activities of reactive microglia and astrocytes associated with prion diseases are dysregulated in opposite directions.</a> Cells. 10 (7), 1728 (2021).</li><li>Stansley, B., Post, J., Hensley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+cell+culture+systems+for+the+study+of+microglial+biology+in+Alzheimer's+disease.">A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.</a> Journal of Neuroinflammation. 9, 115 (2012).</li><li>Shan, Z., 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=Therapeutic+effect+of+autologous+compact+bone-derived+mesenchymal+stem+cell+transplantation+on+prion+disease.">Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease.</a> Journal of General Virology. 98 (10), 2615-2627 (2017).</li><li>Johnson, T. 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Here we demonstrate a reliable and relatively inexpensive protocol for assessing the effects of adipose-derived mesenchymal stromal cells (AdMSCs) in decreasing prion-induced inflammation in a glial cell model. AdMSCs can easily be isolated and expanded in culture for use in as little as 1 week. This protocol consistently produces a heterologous population of cells that express markers consistent with those of mesenchymal stromal cells by immunofluorescence and flow cytometry, and retain immunological function when intro...

Glial inflammation plays a key role in a variety of neurodegenerative diseases, including Parkinson&#39;s, Alzheimer&#39;s, and prion disease. Although abnormal protein aggregation is attributed to much of disease pathogenesis and neurodegeneration, glial cells also play a part in exacerbating this 1,2,3. Therefore, targeting glial-induced inflammation is a promising therapeutic approach. In prion disease, the cellular prion protein (PrPC) misfolds to the disease-associated prion protein (PrPSc), which forms oligomers and aggregates and disrupts homeostasis in the brain 4,5,6.
One of the earliest signs of prion disease is an inflammatory response from astrocytes and microglia. Studies suppressing this response, either by removal of microglia or modification of astrocytes, have generally shown no improvement on, or worsened, disease pathogenesis in animal models 7,8,9. Modulating glial inflammation without eliminating it is an intriguing alternative as a therapeutic.
Mesenchymal stromal cells (MSCs) have taken the stage as a treatment for a variety of inflammatory diseases, due to their ability to modulate inflammation in a paracrine manner 10,11. They have shown the ability to migrate to sites of inflammation and respond to signaling molecules in these environments by secreting anti-inflammatory molecules, growth factors, microRNAs, and more 10,12,13. We have previously demonstrated that MSCs derived from adipose tissue (denoted AdMSCs) are able to migrate toward prion-infected brain homogenate and respond to this brain homogenate by upregulating gene expression for anti-inflammatory cytokines and growth factors.
Moreover, AdMSCs can decrease the expression of genes associated with Nuclear Factor-kappa B (NF-&#954;B), the Nod-Like Receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling, and glial activation, in both BV2 microglia and primary mixed glia 14. Here, we provide protocols on how to isolate both AdMSCs and primary mixed glia from mice, stimulate AdMSCs to upregulate modulatory genes, assess AdMSC migration, and co-culture AdMSCs with prion-infected glia. We hope that these procedures can provide a foundation for further investigation of the role of MSCs in regulating glial-induced inflammation in neurodegenerative and other diseases.

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		<tr>
			<td>0.25% Trypsin</td>
			<td>Cytiva</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipets</td>
			<td>Celltreat</td>
			<td>229005B</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dishes</td>
			<td>Peak Serum</td>
			<td>PS-4002</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml serological pipets</td>
			<td>Celltreat</td>
			<td>229210</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Celltreat</td>
			<td>667015B</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Celltreat</td>
			<td>667050B</td>
			<td></td>
		</tr>
		<tr>
			<td>BV2 microglia cell line</td>
			<td>AcceGen Biotech</td>
			<td>ABC-TC212S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Biologix Research Company</td>
			<td>70-2180</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Electron Microscopy Sciences</td>
			<td>&#160;12785</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Thermo Scientific</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Caisson Labs</td>
			<td>DFL14-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase-I</td>
			<td>Sigma Aldrich</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential amino acids</td>
			<td>Thermo Scientific</td>
			<td>11130051</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>EMD Millipore</td>
			<td>EX0276-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (heat inactivated)</td>
			<td>Peak Serum</td>
			<td>PS-FB4</td>
			<td>Can be purchased as heat inactivated or inactivated in the laboratory</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>EMD Millipore</td>
			<td>1.04003.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass 10 mL serological pipet</td>
			<td>Corning</td>
			<td>&#160;7077-10N</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balances Salt Solution</td>
			<td>Sigma Aldrich</td>
			<td>H8264-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer/Neubauer Chamber</td>
			<td>Daigger</td>
			<td>HU-3100</td>
			<td></td>
		</tr>
		<tr>
			<td>High Glucose DMEM</td>
			<td>Cytiva</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>low glucose DMEM containing L-glutamine</td>
			<td>Cytiva</td>
			<td>SH30021.01</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM/EBSS</td>
			<td>Cytiva</td>
			<td>SH30024.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>non-essential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7145-100M</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16%)</td>
			<td>MP Biomedicals</td>
			<td>219998320</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/neomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4083-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Cytiva</td>
			<td>&#160;SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IFN-gamma Protein</td>
			<td>R&#38;D Systems</td>
			<td>485-MI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse TNF-alpha (aa 80-235) Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>410-MT</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote</td>
			<td>Sigma Aldrich</td>
			<td>SL2-100ML</td>
			<td>Coat inside of glass pipets by aspirating up and down twice in Sigmacote and allowing to dry thoroughly. Wrap in aluminum foil and autoclave pipets 24 h later.</td>
		</tr>
		<tr>
			<td>Stemxyme</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS004106</td>
			<td>Collagenase/Dispase mixture</td>
		</tr>
		<tr>
			<td>Sterile, individually wrapped cotton swab</td>
			<td>Puritan Medical</td>
			<td>&#160;25-8061WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Thincert Tissue Culture Inserts, 24 well, Pore Size=8 &#181;m</td>
			<td>Greiner Bio-One</td>
			<td>662638</td>
			<td></td>
		</tr>
		<tr>
			<td>Thincert Tissue Culture Inserts, 6 well, Pore Size=0.4 &#181;m</td>
			<td>Greiner Bio-One</td>
			<td>657641</td>
			<td></td>
		</tr>
	</tbody>
</table>

adipose-derived mesenchymal stromal cells co-cultured with primary mixed glia to reduce prion-induced inflammation

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments.
One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation.
A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Author Spotlight: Insight Into Advances in Prion Diseases Research

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation

Our work focuses on prion diseases, which are fatal neurodegenerative diseases caused by the misfolding of the prion protein in the brain. Few studies have focused on the impact of glial cells on neuronal death. We want to understand if halting inflammation caused by glial cells can be a therapeutic approach.There has been a recent discovery in the mesenchymal stromal cell field, revealing that the protective properties stem from the extracellular vesicles they release. We aim to delve deeper into the contents of these vesicles and understand how they provide protection against prion-induced glial inflammation. Currently in the field of neurodegeneration, there are a few avenues that are successful at halting neuroinflammation.These include small molecules and genetic modulation using adeno or lentiviruses. However, none have been able to fully reduce inflammation, specifically in large areas of the brain. The most significant finding that we have been able to establish is that through cell therapy, we are able to slow prion-disease progression through inhibition of glial inflammation.The use of a combined with the primary glial-cell model of disease will allow us to continue to ask about cellular stress pathways important in neuroprotection, in a more efficient and direct matter than the use of solely in-vivo models.

Stimulating AdMSCs with TNF&#945; or interferon-gamma (IFN&#947;) for 24 h induces changes in the expression of anti-inflammatory molecules and growth factors. Treating AdMSCs with TNF&#945; or interferon-gamma (IFN&#947;) increases TNF-stimulated gene 6 (TSG-6) mRNA, whereas TNF&#945;, but not IFN&#947;, causes an increase in transforming growth factor beta-1 (TGF&#946;-1)&#160;mRNA. Stimulation with TNF&#945; or IFN&#947; induces an increase in vascular endothelial growth factor (VEGF)mRNA, but no cha...

Watch this Scientific Journal Video about Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation at JoVE.com

Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth ...

adipose-derived-mesenchymal-stromal-cells-co-cultured-with-primary

Research

JoVE Journal

Neuroscience

302 ビュー.  Colorado State University. 私たちの研究は、脳内のプリオンタンパク質のミスフォールディングによって引き起こされる致命的な神経変性疾患であるプリオン病に焦点を当てています。グリア細胞が神経細胞死に与える影響に焦点を当てた研究はほとんどありません。私たちは、グリア細胞によって引き起こされる炎症を止めることが治療アプローチであるかどうかを理解したいと考えています?...

ビデオ: 著者スポットライト:プリオン病研究の進歩への洞察

Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

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automated impactor for contusive spinal cord injury model in mice

Author Spotlight: Insight Into Innovations in Spinal Cord Injury Research

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying...

A Spinal Cord Injury Contusion Device for Accurate Mouse Models

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visualizing monocarboxylates and other relevant metabolites in the ex vivo drosophila larval brain using genetically encoded sensors

Author Spotlight: Advances in Brain Energy Metabolism Research Using the &lt;em&gt;Drosophila&lt;/em&gt; Model

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain...

Monitoring Brain Metabolite Transport in &lt;em&gt;Drosophila&lt;/em&gt; Using FRET Sensors

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electric cell-substrate sensing for real-time evaluation of metal-organic framework toxicological profiles

Author Spotlight: Advances in Evaluating Human Lung Epithelial Cells&#039; Response to Metal-Organic Frameworks 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Evaluating MOF-Induced Cell Behavior Changes by Electric Cell-Substrate Sensing

swine models of aneurysmal diseases for training and research

Swine Models of Aneurysmal Diseases for Training and Research

Description of two different aneurysmal swine models for neuroradiology training courses and research studies. This study provides evidence of the feasibility of these aneurysm porcine model creations and the reproducible methods that are close to the clinical...

using avian skin explants to study tissue patterning and organogenesis

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

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detection of abnormal prion protein by immunohistochemistry

Detection of Abnormal Prion Protein by Immunohistochemistry

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected...

orienteering as a tool for cognitive research: an implementation guide

Author Spotlight: Utilizing Orienteering in Research to Improve Brain Health and Spatial Memory

The sport of orienteering is emerging as an effective way to train both the body and brain as it combines physical activity with spatial navigation. The current manuscript offers a guide on how to implement the sport of orienteering in...

Implementing Orienteering for Body and Brain Training 

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mechanistic insight into the development of tnbs-mediated intestinal fibrosis and evaluating the inhibitory effects of rapamycin

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal...

hepatic glucose production, ureagenesis, and lipolysis quantified using the perfused mouse liver model

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic...

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

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fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic...

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

murine left pulmonary hilar clamp model of lung ischemia reperfusion injury

Author Spotlight: Insights into the Effect of Ischemia Reperfusion on Lung Transplantation

The protocol outlines a feasible, reliable, and reproducible method of left pulmonary hilar clamping that can be used to study lung ischemia-reperfusion injury in mouse...

Mouse Model for Studying Lung Ischemia-Reperfusion Injury

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local hyperthermia for warts treatment

Author Spotlight: Dermatopathology and the Treatment of Sexually Transmitted Diseases

Here, we present a protocol for local hyperthermia, a new and effective therapeutic for treating warts. We also showcase its safety and efficacy as an independent...

Effective Wart Treatment with Local Hyperthermia: A Minimally Invasive Approach

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functional magnetic resonance imaging (fmri) of the visual cortex with wide-view retinotopic stimulation

Author Spotlight: Insights into Visual Cortex Research Through Wide-View fMRI Mapping 

We have developed techniques for mapping the visual cortex function utilizing more of the visual field than is commonly used. This approach has the potential to enhance the evaluation of vision disorders and eye...

Advancing Visual Cortex Mapping for Enhanced Evaluation of Vision Disorders

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use of micropipette-guided drug administration as an alternative method to oral gavage in rodent models

Author Spotlight: Advancing Animal Research with Less Invasive Drug Administration

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

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corneal sensitivity testing procedure for ophthalmologic and optometric patients

 Author Spotlight: Establishing a Practical and Cost-Effective Protocol for Corneal Sensitivity Testing in Clinical Settings

Corneal sensitivity testing provides insight into the health of the corneal nerves and helps diagnose ocular surface diseases. We present a concise protocol to qualitatively assess corneal sensitivity that can be readily used by eye care providers across clinical settings.

Assessing the Health of the Corneal Nerves in Ocular Surface Diseases

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detecting abnormal prion proteins in brain tissue using immunohistochemistry

This video demonstrates a technique to detect misfolded prion protein in brain sections using immunohistochemistry. Upon treating the brain section with formic acid to denature prions and minimize infection risk, as well as unmasking the prion aggregates using heat-induced epitope retrieval, the sections are immunolabeled for the misfolded prion protein aggregates and observed under a...

Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry

methodology to metabolically inactivate bacteria for caenorhabditis elegans research

Author Spotlight: Advancing Caenorhabditis elegans Research Using Paraformaldehyde-Treated Bacteria 

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described...

Bacterial Metabolic Inactivation in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt; Using Paraformaldehyde

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advances in nanoscale infrared spectroscopy to explore multiphase polymeric systems

Author Spotlight: Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems  

This protocol describes the application of atomic force microscopy and nanoscale infrared spectroscopy to evaluate the performance of photothermal nanoscale infrared spectroscopy in the characterization of three-dimensional multi-polymeric samples.

Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems

ginger moxibustion, a non-pharmacological treatment, for diarrhea in patients with chronic obstructive pulmonary disease

Author Spotlight: Exploring Non-Pharmacological Therapies for Chronic Respiratory Diseases &#8212; Linking Intestinal Microbiome Insights to COPD Treatment

Here, we present the method of using ginger moxibustion to treat diarrhea in patients with chronic obstructive pulmonary...

Assessing the Efficacy of Ginger Moxibustion in Diarrhea Management in COPD

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Isolating and Infecting Primary Cortical Mixed Glia with Prions

To begin, take the brain isolated from the euthanized mouse pups and place it in a three-centimeter Petri dish containing cold MEM with EBSS and 2X PSN. Under a dissecting scope, separate brain hemispheres, remove and discard the midbrain, hippocampus, and meninges. Place both cortical hemispheres in a 50-milliliter conical tube containing five milliliters of MEM EBSS with 2X PSN, and keep on ice.In a tissue culture hood, aspirate the media from the 50-milliliter conical tube using a pipette, leaving the cortical tissue pieces at the bottom. Add 10 milliliters of pre-warmed dissociation media with a coated glass pipette, and triturate the tissue 10 to 20 times. Transfer the suspension to a 50-milliliter beaker containing a small stir bar and place it on a stir plate.Then remove the beaker, set it at a 30-degree angle for three minutes to allow the tissue to settle, and transfer the cell suspension to a new 50-milliliter conical tube on ice. Centrifuge the 50-milliliter conical tube at 1000G for 10 minutes at four degrees Celsius. Replace the supernatant with glial growth medium and count the cells using a hemocytometer.Plate the cells at a density of one million in 10-centimeter cell culture-treated dishes and incubate for 24 hours. Then replace the media with fresh glial media and allow the cells to reach 100%confluence within two weeks before plating them for experimentation. Plate 100, 000 glial cells per well in six-well plates and allow them to reach 80%to 100%confluence for in vitro prion infection.Expose 20%diluted brain homogenates and PBS to ultraviolet light for 30 minutes. Aspirate the media from the Glia to be infected and add 1.5 to 2 milliliters of glial growth media with 0.1%normal or prion brain homogenate per well. After 72 hours, remove the media, wash the cells with PBS and add fresh glial growth media.Carefully aspirate the adipose tissue in approximately one milliliter of HBSS in 0.25%trypsin solution, using a serological pipette. Transfer the tissue to a four-centimeter Petri dish containing two milliliters of DMEM F-12 media with Dnase-1, collagenase and Dispase mixture. Then cut the adipose tissue into small pieces using scissors and incubate the mixture at 37 degrees Celsius for 1.5 hours.Transfer the tissue to a 50-milliliter conical tube, triturate the tissue, and pellet the stromal vascular fraction, which will appear red. After washing the pellet with sterile PBS and centrifuging for three minutes, resuspend the pellet in one milliliter of ADMSC media. Afterward, pipette the cell suspension through a 40-micrometer strainer into a sterile 50-milliliter conical tube, to remove non-dissociated tissue.Add nine milliliters of ADMSC media to a 10-centimeter cell culture-treated dish. Pipette the strained cell suspension into it and incubate at 37 degrees Celsius with 5%carbon dioxide. Change the media the next day and passage the cells once they attain 80%to 90%confluency.After the cells have undergone two to three passages, resuspend them in three to 10 milliliters of media and count using a hemocytometer. Plate 100, 000 cells per well in six-well plates containing ADMSC media and incubate overnight, as shown earlier. The next day, prepare the ADMSC media for cytokine-stimulated cells with either 10 nanograms per milliliter of TNF-alpha or 200 nanograms per milliliter of IFN-gamma.Create ADMSC media with a final concentration of 0.1%prion-infected or normal brain homogenate for control samples. Aspirate the old media, add 1.5 milliliters of cytokine or brain homogenate-containing media into the corresponding wells in triplicate, and return plates to the incubator. Then wash the cells with PBS and isolate RNA by adding 350 microliters of lysis buffer with 1%beta-mercaptoethanol to each well.Remove cell lysates using a cell lifter and reverse transcribe 25 nanograms of RNA per sample and amplify complementary DNA. Plate BV2 microglia at 50, 000 cells per well or primary mixed glia at 100, 000 cells per well in a six-well plate. Treat BV2 cells with media containing 0.1%prion-infected or normal brain homogenate and incubate for 72 hours.Following incubation, wash the cells two times with PBS to remove the remaining brain homogenate. Add fresh media to the cells and return the plate to the incubator. To stimulate ADMCs, treat them with 10 nanograms per milliliter of TNF-alpha for 24 hours before co-culturing with BV2 or mixed glia.Wash the stimulated ADMCs with PBS three times and spin the ADMSC suspension as demonstrated earlier. Replace the media on BV2 cells or mixed glia with two milliliters per well of ADMSC media, and place inserts for six-well plates with a pore size of 0.4 micrometers into half of the wells. Add two milliliters of ADMSC media to each insert and add an additional two milliliters of media to wells that do not receive inserts.Upon pelleting, resuspend and counting the ADMCs, add 50, 000 to 100, 000 cells to each insert and incubate them. At the end of the incubation, remove and discard the inserts or replace them in a new six-well plate and wash the inserts twice with PBS. Add the buffer provided by the RNA isolation kit to the mixed glia or BV2 cells and scrape the wells.

Migration Assay of Murine Adipose-Derived Mesenchymal Stromal Cells

Treat passage two AdMSCs with media containing 10 nanograms per milliliter of TNF alpha for 24 hours or without TNF alpha for unstimulated cells. After washing and incubating the cells in serum-free media for four hours, add 25, 000 AdMSCs to each insert and incubate for 24 hours at 37 degrees Celsius. Aspirate the contents of both well and insert, then wash twice with PBS leaving one milliliter of PBS in each well.Using forceps, remove the inserts one by one and gently but thoroughly wipe the top chamber with a cotton swab to remove unmigrated cells. Aspirate the remaining PBS and pipette 500 microliters of crystal violet solution to the well and the top chamber of the insert. Aspirate the crystal violet solution from the well and top chamber of the insert after one hour incubation.After washing and wiping the top chamber, place the insert in a new 24 well plate containing one milliliter of PBS. Using an inverted microscope with a camera, image four random areas towards the insert's center under a 10 times objective. Upload the images to the preferred image processing software and adjust the background for enhanced contrast with purple stained cells.CLIA infected with 22L showed increased mRNA expression for inflammatory markers CCL2, CCL5, and IL1 beta, and the astrocyte marker S100 beta compared to those treated with NBH. Coculturing with AdMSCs decreased expression of the inflammatory cytokines CCL2, CCL5, and IL1 beta, but not TNF alpha for both MBH and 22L infected cells. BV2s cocultured with AdMSCs showed a decrease in mRNA for the inflammatory markers IL1 beta, IL-6, TNF alpha, and the complement protein C1qa in both NBH and RML treated cells.Additionally, AdMSCs decreased the M1 microglia gene CD16 and increased the M2 microglia marker ARG-1. The in vitro migration assay suggested that AdMSCs showed significant migration toward media containing 1%RML.