This work was supported by NIH grants HL-089779 (DW), HL-112270 (KAP) and HL-102836 (KAP) and Cimphoni Life Sciences (part of DW salary). The authors thank Meghann Sytsma for editing the manuscript.

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The authors declare that they have no competing financial interests.

目標は、TSK / +マウスの心臓の炎症、線維症、および血管機能にIRF5の役割を解明するためにIRF5阻害剤を設計することでした。知見はIRF5Dの増殖またはアポトーシスを誘導しなかったことです。また、炎症を低減し、血管機能を改善しました。これらのデータは、IRF5はTSK / +マウスの中心部に炎症や線維症の発症に重要な機構的な役割を果たしていることを示唆し、それが治療標的として機能す...

<html>細胞増殖と細胞死の免疫応答の調節は、インターフェロン調節因子の転写因子ファミリーの役割の中心です。 IRF5は、タイプ1の間の免疫応答の調節、炎症促進応答および2型、免疫応答のターゲット組織修復のために重要であると強調表示されます。 IRF5癌1、及び自己免疫2、3、4、5のキーです。 タイト皮膚マウス（TSK / +）は、組織線維症とフィブリリン1遺伝子における重複変異に起因する強皮症のモデルです。タイト皮膚および結合組織の増加でこの変異は結果。これらのマウスは、心筋の炎症、線維化および最終的に心不全5、6、7を開発します&gt; 8,9。強皮症は、米国6で約15万人の患者に影響を与える自己免疫線維性疾患です。この疾患の特徴は、心臓7、8、9、10、11を含む内臓の線維症です。 研究の性質は、阻害ペプチドの設計を要求しました。ソフトウェアアプローチは、ファージディスプレイを使用する従来のアプローチを介して選択しました。ソフトウェアのアプローチが容易になり、より少ない時間のかかるものです。 RCSBのデータバンクは、適切な結合部位12を同定するために使用されます。組換えタンパク質で新たに設計したペプチドの相互作用を研究し、結合パラメーターに焦点を当てて、干渉生物層と呼ばれる技術を使用しました。生物層の干渉は、バイオセンサベースのtechniqですバイオセンサーと結合サンプルを使用して結合親和性、会合及び解離を決定するUE。バイオセンサーは、蛍光、ルミネセンス、放射量および比色標識することができます。測定は、質量付加または枯渇が会合及び解離13、14に似ているに基づいています。本研究の目的は、心筋の炎症や線維症におけるIRF5の役割を理解することでした。目標は、組織線維症および強皮症の開発にIRF5の役割への洞察を得ることでした。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100- 100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-labeled goat anti-mouse IgG antibody&#160;</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bardford reagent</td>
			<td>Thermo Fisher</td>
			<td>23200</td>
			<td>Pierce&#160;</td>
		</tr>
		<tr>
			<td>Biosensors</td>
			<td>Forte-Bio</td>
			<td>MR18-0009</td>
			<td></td>
		</tr>
		<tr>
			<td>CD64 (H-250)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-15364</td>
			<td></td>
		</tr>
		<tr>
			<td>CellEvent Caspase-3/7 Substrate</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>C10427</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter AQueous One Solution Cell Proliferation Assay kit</td>
			<td>Promega</td>
			<td>G3580</td>
			<td>Promega</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher</td>
			<td>D-1306</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>donkey anti rat Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-21208</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>ECL plus</td>
			<td>GE healthcare/Amersham</td>
			<td>RPN2133</td>
			<td>After a lot of trial and error we came back to this one</td>
		</tr>
		<tr>
			<td>Eclipse TE 200-U microscope with EZ C1 laser scanning software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-11008</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>HRP&#160; anti-goat</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-516086</td>
			<td>!:10000 dilution in TBS</td>
		</tr>
		<tr>
			<td>HRP donkey anti-mouse</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-2315</td>
			<td>1:10000 dilution in TBS</td>
		</tr>
		<tr>
			<td>ICAM-1 antibody</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-1511</td>
			<td>1:200 dilution in PBS</td>
		</tr>
		<tr>
			<td>IRF5 antibody (H56)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-98651</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro plate reader Elx800</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIMP neutrophil marker</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-133821</td>
			<td>1:200 dilution in PBS</td>
		</tr>
		<tr>
			<td>Octet RED</td>
			<td>Forte Bio</td>
			<td></td>
			<td>protein-protein binding</td>
		</tr>
		<tr>
			<td>Peptide design&#160; Medit SA software</td>
			<td>RCSB.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant IRF5 protein synthesis</td>
			<td>TopGene Technologies</td>
			<td></td>
			<td>protein expression, synthesis service</td>
		</tr>
		<tr>
			<td>sodium dodecyl phosphate</td>
			<td>Sigma Aldrich</td>
			<td>436143</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pharmacy</td>
			<td></td>
			<td>Schedule III controlled substance, presciption required&#160;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>MedVet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5X-45X Trinocular Dissecting Zoom Stereo Microscope with Gooseneck LED Lights</td>
			<td>Am Scope</td>
			<td>SKU: SM-1TSX-L6W</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Desalting Columns</td>
			<td>Thermofisher</td>
			<td>2161515</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Media EBM Bullet kit</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td>kit contains growth supplemets</td>
		</tr>
		<tr>
			<td>VIA-100K</td>
			<td>&#160;Boeckeler Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-15% TGX gel</td>
			<td>Bio-Rad</td>
			<td>5671081</td>
			<td></td>
		</tr>
		<tr>
			<td>MedSuMo software</td>
			<td>Medit, Palaiseau, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli Buffer</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

図1で実証結果は、ペプチドを設計する方法を示しています。 図1、左上には、多くのキナーゼによってリン酸化されるIRF5中の領域（2黄色の矢印の間、アミノ酸（AA）425から436）を示しています。 図1、右上は、IRF5のリン酸化されたドメインが結合黄色の楕円形を示しています。 3DSHの二量体構造は、ヘリックス2（aa303-312）の左?...

Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

単離された血管の血管拡張およびタイト皮膚マウスの細胞外マトリックスの単離

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

vasodilation-isolated-vessels-isolation-extracellular-matrix-tight

Research

JoVE Journal

Immunology and Infection

8.0K ビュー.  Medical College of Wisconsin. We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide. 

ビデオ: Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJLマウスにおける実験的自己免疫性下垂体炎の誘導

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

免疫・感染学

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脳から隔離された白血球の単離と解析<em&gt;マラリアベルゲイ</em&gt; ANKA感染マウス

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

離とナイーブCD4 Tリンパ球のTh17分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

マウスにおけるローカルアナフィラキシーの測定

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

肥満に関連する炎症によって影響を受ける組織のマクロファージの精製と性質、分離

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

付着性のセルの細菌感染症に及ぼす細胞外マトリックス剛性を勉強するためのウェル フォーマット ポリアクリルアミドを用いたアッセイ

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

単離された血管の血管拡張およびタイト皮膚マウスの細胞外マトリックスの単離

要約

さらに動画を探す

この動画の章

SJLマウスにおける実験的自己免疫性下垂体炎の誘導

細菌付着の研究ではせん断応力を導入する

脳から隔離された白血球の単離と解析

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

離とナイーブCD4 Tリンパ球のTh17分化

マウスにおけるローカルアナフィラキシーの測定

肥満に関連する炎症によって影響を受ける組織のマクロファージの精製と性質、分離

付着性のセルの細菌感染症に及ぼす細胞外マトリックス剛性を勉強するためのウェルフォーマットポリアクリルアミドを用いたアッセイ

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

心の血管外免疫細胞の分離と同定

単離された血管の血管拡張およびタイト皮膚マウスの細胞外マトリックスの単離

要約

さらに動画を探す

この動画の章

SJLマウスにおける実験的自己免疫性下垂体炎の誘導

細菌付着の研究ではせん断応力を導入する

脳から隔離された白血球の単離と解析

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

離とナイーブCD4 Tリンパ球のTh17分化

マウスにおけるローカルアナフィラキシーの測定

肥満に関連する炎症によって影響を受ける組織のマクロファージの精製と性質、分離

付着性のセルの細菌感染症に及ぼす細胞外マトリックス剛性を勉強するためのウェル フォーマット ポリアクリルアミドを用いたアッセイ

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

心の血管外免疫細胞の分離と同定

付着性のセルの細菌感染症に及ぼす細胞外マトリックス剛性を勉強するためのウェルフォーマットポリアクリルアミドを用いたアッセイ