<meta charset="utf8"></head><body>臍帯血および骨髄からのHSPCを使用した赤血球生成のモデリング

<ol>
	<li>Sender, R., Milo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+cellular+turnover+in+the+human+body.">The distribution of cellular turnover in the human body.</a> Nat Med. 27 (1), 45-48 (2021).</li><li>Cazzola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ineffective+erythropoiesis+and+its+treatment.">Ineffective erythropoiesis and its treatment.</a> Blood. 139 (16), 2460-2470 (2022).</li><li>Silverberg, D. S., Iaina, A., Wexler, D., Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathological+consequences+of+anaemia.">The pathological consequences of anaemia.</a> Clin Lab Haematol. 23 (1), 1-6 (2001).</li><li>Safiri, S., 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=Burden+of+anemia+and+its+underlying+causes+in+204+countries+and+territories,+1990-2019:+results+from+the+Global+Burden+of+Disease+Study+2019.">Burden of anemia and its underlying causes in 204 countries and territories, 1990-2019: results from the Global Burden of Disease Study 2019.</a> J Hematol Oncol. 14 (1), 185 (2021).</li><li>Bondu, S., 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=A+variant+erythroferrone+disrupts+iron+homeostasis+in+SF3B1-mutated+myelodysplastic+syndrome.">A variant erythroferrone disrupts iron homeostasis in SF3B1-mutated myelodysplastic syndrome.</a> Sci Transl Med. 11 (500), eaav5467 (2019).</li><li>Elvarsdottir, E. 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=A+three-dimensional+in+vitro+model+of+erythropoiesis+recapitulates+erythroid+failure+in+myelodysplastic+syndromes.">A three-dimensional in vitro model of erythropoiesis recapitulates erythroid failure in myelodysplastic syndromes.</a> Leukemia. 34 (1), 271-282 (2020).</li><li>Yip, B. 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=Effects+of+L-leucine+in+5q-+syndrome+and+other+RPS14-deficient+erythroblasts.">Effects of L-leucine in 5q- syndrome and other RPS14-deficient erythroblasts.</a> Leukemia. 26 (9), 2154-2158 (2012).</li><li>Clough, C. 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=Coordinated+missplicing+of+TMEM14C+and+ABCB7+causes+ring+sideroblast+formation+in+SF3B1-mutant+myelodysplastic+syndrome.">Coordinated missplicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome.</a> Blood. 139 (13), 2038-2049 (2022).</li><li>Chao, R., Gong, X., Wang, L., Wang, P., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD71(high)+population+represents+primitive+erythroblasts+derived+from+mouse+embryonic+stem+cells.">CD71(high) population represents primitive erythroblasts derived from mouse embryonic stem cells.</a> Stem Cell Res. 14 (1), 30-38 (2015).</li><li>Loken, M. R., Shah, V. O., Dattilio, K. L., Civin, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+human+bone+marrow:+I.+Normal+erythroid+development.">Flow cytometric analysis of human bone marrow: I. Normal erythroid development.</a> Blood. 69 (1), 255-263 (1987).</li><li>Marsee, D. K., Pinkus, G. S., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD71+(transferrin+receptor):+an+effective+marker+for+erythroid+precursors+in+bone+marrow+biopsy+specimens.">CD71 (transferrin receptor): an effective marker for erythroid precursors in bone marrow biopsy specimens.</a> Am J Clin Pathol. 134 (3), 429-435 (2010).</li><li>Berastegui, N., 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+transcription+factor+DDIT3+is+a+potential+driver+of+dyserythropoiesis+in+myelodysplastic+syndromes.">The transcription factor DDIT3 is a potential driver of dyserythropoiesis in myelodysplastic syndromes.</a> Nat Commun. 13 (1), 7619 (2022).</li><li>Mian, S. 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=Vitamin+B5+and+succinyl-CoA+improve+ineffective+erythropoiesis+in+SF3B1-mutated+myelodysplasia.">Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.</a> Sci Transl Med. 15 (685), eabn5135 (2023).</li><li>Armes, 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=Germline+ERCC+excision+repair+6+like+2+(ERCC6L2)+mutations+lead+to+impaired+erythropoiesis+and+reshaping+of+the+bone+marrow+microenvironment.">Germline ERCC excision repair 6 like 2 (ERCC6L2) mutations lead to impaired erythropoiesis and reshaping of the bone marrow microenvironment.</a> Br J Haematol. 199 (5), 754-764 (2022).</li><li>Hug, N., 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=A+dual+role+for+the+RNA+helicase+DHX34+in+NMD+and+pre-mRNA+splicing+and+its+function+in+hematopoietic+differentiation.">A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation.</a> RNA. 28 (9), 1224-1238 (2022).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2x HEPES</td>
			<td>Merck</td>
			<td>51558</td>
			<td></td>
		</tr>
		<tr>
			<td>Beads</td>
			<td>Thermo Scientific</td>
			<td>01-2222-42</td>
			<td></td>
		</tr>
		<tr>
			<td>CD235a APC Cy7</td>
			<td>Biolegend</td>
			<td>349115</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 PerCP Cy5.5 antibody</td>
			<td>BD Biosciences</td>
			<td>347222</td>
			<td></td>
		</tr>
		<tr>
			<td>CD71 PE antibody</td>
			<td>Biolegend</td>
			<td>334106</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Merck</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Merck</td>
			<td>D4527-200KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mounting medium&#160;</td>
			<td>VWR</td>
			<td>1.00579.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Fusion&#160;</td>
			<td>BD Biosciences</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Merck</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Merck</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa solution</td>
			<td>Abcam</td>
			<td>ab150670</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Merck</td>
			<td>H0887-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EPO</td>
			<td>PeproTech</td>
			<td>100-64</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Flt-3 ligand</td>
			<td>PeproTech</td>
			<td>300-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Human G-CSF</td>
			<td>PeproTech</td>
			<td>300-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IGF1</td>
			<td>PeproTech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6</td>
			<td>PeproTech</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Human SCF</td>
			<td>PeproTech</td>
			<td>300-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TPO</td>
			<td>PeproTech</td>
			<td>AF-300-18</td>
			<td></td>
		</tr>
		<tr>
			<td>LSRFortessa Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>May-Grunwald solution&#160;</td>
			<td>Generon</td>
			<td>26250-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pannoramic 250 High Throughput Scanner&#160;</td>
			<td>3DHISTECH</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Cytospin 3&#160;</td>
			<td>Thermo Scientific</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Medium medium</td>
			<td>Stem Cell Technologies</td>
			<td>9655</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive pipeline to assess the efficiency of human erythropoiesis in vitro and ex vivo

<meta charset="utf8"></head><body>著者スポットライト:赤血球生成研究の進歩 - 骨髄異形成症候群における造血幹細胞機能の評価のための簡素化されたパイプライン

ヒト赤血球生成の効率をin vitroおよびex vivoで評価するための包括的なパイプライン

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 ...

a-comprehensive-pipeline-to-assess-efficiency-human-erythropoiesis

Research

JoVE Journal

Biology

A Comprehensive Pipeline to Assess the Efficiency of Human Erythropoiesis In Vitro and Ex Vivo

324 ビュー.  INSERM U1242, University of Rennes. 赤血球生成のモデリングは、このプロセスにおけるアクターの生物学的意義を理解し、分化欠陥を緩和する可能性のある新しい治療アプローチを評価する貴重な機会を提供します。ここでは、ex vivoでCD34+造血幹細胞と前駆細胞を効率的に分化するためのシンプルで信頼性の高い方法について説明します。

ビデオ: 著者スポットライト:赤血球生成研究の進歩 - 骨髄異形成症候群における造血幹細胞機能の評価のための簡素化されたパイプライン

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

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

生物学

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

初代ヒト卵管上皮細胞のex vivoでの文化

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

ex vivoで培養マウス胚の皮膚およびメラノブラストの移行のライブイメージング

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

SCAPの分析<em&gt; N</em&gt;ヒト細胞における-glycosylationと人身売買

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

子宮内電散法における両側性を用いたパイプラインによるげっ歯類の行動に対する遺伝的影響の調査

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...