<meta charset="utf8"></head><body>蛍光顕微鏡分析のための肺における真菌胞子局在の保存(英語)

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Club+cell+TRPV4+serves+as+a+damage+sensor+driving+lung+allergic+inflammation.">Club cell TRPV4 serves as a damage sensor driving lung allergic inflammation.</a> Cell Host Microbe. 27 (4), 614-628.e6 (2020).</li><li>Evans, S. E., Hahn, P. Y., McCann, F., Kottom, T. J., Pavlovic', Z. V., Limper, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumocystis+cell+wall+&#946;-glucans+stimulate+alveolar+epithelial+cell+chemokine+generation+through+nuclear+factor-&#954;B-dependent+mechanisms.">Pneumocystis cell wall &#946;-glucans stimulate alveolar epithelial cell chemokine generation through nuclear factor-&#954;B-dependent mechanisms.</a> Am J Respir Cell Mol Biol. 32 (6), 490-497 (2005).</li><li>Bauer, C., Krueger, M., Lamm, W. J. E., Glenny, R. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+and+inflation+of+the+mouse+lung+for+tumor+histology.">Perfusion and inflation of the mouse lung for tumor histology.</a> J Vis Exp. 162, e60605 (2020).</li><li>Hsia, C. C. W., Hyde, D. M., Ochs, M., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+Standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: Standards for quantitative assessment of lung structure.</a> Am J Respir Crit Care Med. 181 (4), 394-418 (2010).</li><li>Gil, J., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+lining+of+bronchioles+after+perfusion-fixation+of+rat+lungs+for+electron+microscopy.">Extracellular lining of bronchioles after perfusion-fixation of rat lungs for electron microscopy.</a> Anat Rec. 169 (2), 185-199 (1971).</li><li>Bachofen, H., Ammann, A., Wangensteen, D., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+fixation+of+lungs+for+structure-function+analysis:+credits+and+limitations.">Perfusion fixation of lungs for structure-function analysis: credits and limitations.</a> J Appl Physiol Respir Environ Exerc Physiol. 53 (2), 528-533 (1982).</li><li>Evans, C. M., 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=The+polymeric+mucin+Muc5ac+is+required+for+allergic+airway+hyperreactivity.">The polymeric mucin Muc5ac is required for allergic airway hyperreactivity.</a> Nat Commun. 6, 6281 (2015).</li><li>Galati, D. F., Asai, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy.">Immunofluorescence microscopy.</a> Curr Protoc. 3 (8), e842 (2023).</li><li>Hickey, S. M., 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=Fluorescence+microscopy-An+outline+of+hardware,+biological+handling,+and+fluorophore+considerations.">Fluorescence microscopy-An outline of hardware, biological handling, and fluorophore considerations.</a> Cells. 11 (1), 35 (2021).</li><li>Risling, T. E., Caulkett, N. A., Florence, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-drop+anesthesia+for+small+laboratory+animals.">Open-drop anesthesia for small laboratory animals.</a> Can Vet J. 53 (3), 299-302 (2012).</li><li>Bodnar, M. J., Ratuski, A. S., Weary, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+isoflurane+anesthesia+using+the+drop+method.">Mouse isoflurane anesthesia using the drop method.</a> Lab Anim. 57 (6), 623-630 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G, 1 1/2 needle (305185)</td>
			<td>Fisher</td>
			<td>305185</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L&#160; Screwtop Jar&#160; (Nalgene)</td>
			<td>Fisher Scientific</td>
			<td>11-823-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Air-Tite Bulk Unsterile Syringes 10 mL Luer Lock</td>
			<td>Fisher</td>
			<td>14-817-175</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSed Injection (xylazine sterile solution)</td>
			<td>Akorn</td>
			<td>59399-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Blocker and Diluent, R.T.U.</td>
			<td>Vector</td>
			<td>SP-5035-100</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Insyte Autoguard Winged Shielded IV Catheter with BD Vialon Catheter Material 18 G x 1.88 in</td>
			<td>BD</td>
			<td>381547</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block&#8482;)</td>
			<td>BD</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Orange CMRA Dye</td>
			<td>Fisher Scientific</td>
			<td>NC0873640</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Labviva</td>
			<td>75003</td>
			<td></td>
		</tr>
		<tr>
			<td>Coccidioides posadasii cts2/ard1/cts3&#916;&#160;</td>
			<td>BEI Resources</td>
			<td>NR-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 70 micron strainers</td>
			<td>VWR</td>
			<td>10054-456</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM AlexaFluor647 monoclonal antibody</td>
			<td>Biolegend</td>
			<td>118211</td>
			<td></td>
		</tr>
		<tr>
			<td>Exel International HYpodermic Needles 30 G x 1/2&#34;</td>
			<td>Labviva</td>
			<td>EN3012</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Syringes for Single Use (1mL, Leur Slip)</td>
			<td>Fisher</td>
			<td>14-955-462</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Monohydrate</td>
			<td>Azer Scientific&#160;</td>
			<td>ES17530-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>High Vacuum Grease</td>
			<td>VWR</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 Solution 20 mM (5 mL)</td>
			<td>ThermoFischer</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane USP</td>
			<td>Covetrus</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Dechra</td>
			<td>1000001250</td>
			<td></td>
		</tr>
		<tr>
			<td>KIMWIPES Delicate Task Wipers (4.4&#39;&#39; x 8.4&#34;)</td>
			<td>VWR</td>
			<td>21905-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Lexer Baby Scissors</td>
			<td>FST</td>
			<td>14078-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>FST</td>
			<td>11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Buffered Formalin (10%) (Azer Scientific)</td>
			<td>Fisher</td>
			<td>22-026-350</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc EasYFlask tissue culture flasks, T75, filter caps</td>
			<td>VWR</td>
			<td>15708-134</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>VWR</td>
			<td>45000-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way embedding molds</td>
			<td>Sigma</td>
			<td>E6032-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>QuPath 0.5.1 Software</td>
			<td>Open-source</td>
			<td>https://qupath.github.io/</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Suture thread size 3/0</td>
			<td>FST</td>
			<td>18020-30</td>
			<td></td>
		</tr>
		<tr>
			<td>SlidesMicro Slides Superfrost Plus&#160;</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFade Glass Soft-set Antifade Mountant (2 mL)</td>
			<td>ThermoFischer</td>
			<td>S36917</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound, Sakura Finetek</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>ThermoFischer</td>
			<td>J20605.AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Vector Laboratories ImmEDGE Hydrophobic Barrier Pen Set Of 2</td>
			<td>Fisher Scientific</td>
			<td>NC9545623</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Micro Cover Glasses, Rectangular (24 mm x 40 mm #1.5)</td>
			<td>VWR</td>
			<td>48393-230</td>
			<td></td>
		</tr>
		<tr>
			<td>White Plastic Wire Mesh</td>
			<td>MAPORCH</td>
			<td>789862904922</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher</td>
			<td>BP9727-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioScan 7&#160;</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>https://www.zeiss.com/microscopy/us/products/imaging-systems/axioscan-for-biology.html</td>
			<td>multichannel fluorescent microscope</td>
		</tr>
		<tr>
			<td>ZEN 3.7 Software</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>https://www.zeiss.com/microscopy/us/products/software/zeiss-zen.html</td>
			<td>microscopy software</td>
		</tr>
	</tbody>
</table>

efficient method for imaging murine lungs that preserves spatial dynamics of fungal spores in the airways

<meta charset="utf8"></head><body>気道内の真菌胞子の空間動態を保存するマウス肺の効率的なイメージング法

気道内の真菌胞子の空間動態を保存するマウス肺の効率的なイメージング法

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

efficient-method-for-imaging-murine-lungs-that-preserves-spatial

Research

JoVE Journal

Immunology and Infection

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

218 ビュー.  Rutgers - The State University of New Jersey. 蛍光顕微鏡による分析のために、肺気道内の真菌胞子の自然な位置を保持する真菌病因モデルの方法を提示します。 

ビデオ: 気道内の真菌胞子の空間動態を保存するマウス肺の効率的なイメージング法

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

免疫・感染学

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

ダルババンシンの参照ブロス微量希釈法<em&gt;インビトロ</em好気的に成長する細菌の&gt;感受性試験

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

マウスリンパ球標識による<sup&gt; 64</sup以下のためのターゲット設定&gt;のCu-抗体の受容体<em&gt;インビボ</em&gt; PET / CTによる細胞輸送

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

炎症性細胞の浸潤を分析するための肺胞の気管支肺胞洗浄

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

に応答したヒト一次好中球及び単球による抗カビ活動のライブイメージング<em&gt; A。フミガーツス</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

マウス小腸における上皮内リンパ球の分体イメージング

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

インビトロ転写mRNAによる一次マクロファージの高効率トランスフェクション

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

マウスにおける神経免疫ダイナミクスの反復インビボイメージングを行う正確な脳マッピング

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

マウス真菌敗血症モデルにおける効率的な尾静脈注射のための現代的な温暖化/抑制装置

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

ヒト胚性幹細胞からの肝細胞様細胞誘導の効率的な方法

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...