NIHの免疫学研修グラントT32 AI 60573（KRG）とNIHの助成金GM 41514（MDC）

前の実験の開始へ： <ul><li> IACUC承認を得る</li><li>手術器具を滅菌し、Q -のヒント</li></ul> I.ハーベストドナー組織<ol><li> （金融機関のSOPに従った）ドナーマウスを安楽死させる</li><li>アルコールと耳の根元から髪をぬらす</li><li>ベースで耳をカットすることによって収穫のドナーの皮膚</li><li>氷の上にプレートの組織を配置</li><li>スプリットの耳：ラットの歯の鉗子との耳のベースで組織のピンチ腹側と背側の両側を持ち、ゆっくりと組織を離れて引っ張る。float型の背側組織、内部の表面の下、PBSで氷の上に、コラーゲン腹フラップを破棄</li></ol> II。移植のための受信者を準備する<ol><li>承認された手順を使用してマウスを麻酔</li><li>術後鎮痛のために鎮痛剤を投与する（すなわち、0.05〜0.1 mg / gのBupernexのIP） </li><li>横方向の位置に配置してマウス</li><li>動物が十分に鎮静されていることを確認するためにつま先のピンチを実行</li><li>濡れたアルコールと髪とひげをそる胸部</li><li>アルコール綿棒できれいな部分を拭いてください</li></ol> III。皮膚移植<ol><li>ラット歯鉗子でテントの皮膚と鉗子以下〜0.25〜0.5センチメートルをカット：〜1cmの切開を行う</li><li> PBS、移植床の上の場所（移植片のベッドが直面している耳の皮膚の内部表面を持つ）から、ドナーの皮膚を削除し、滅菌綿棒で余分なPBSを除去</li><li>その移植は、移植床（片エッジが下に丸まっていないとその移植片は、受信者のトランクの皮膚への二日酔いをしないしていることを確認して）の中できっぱりとあるので、慎重に過剰ドナーの皮膚を切り落とす</li><li>無菌包帯でラップの受信者（皮膚移植の上に置く非粘着ガーゼセグメントで） </li><li>その包帯が（動物の呼吸を評価する）あまりにもタイトではないことを確認し、その腕は自由に携帯電話です。 </li><li>単一の2から0絹縫合糸による安全な包帯</li><li>清潔なケージでの場所の動物（加熱ランプの下の動物まで、十分に麻酔から回復した） </li><li>モニター受信者苦痛の徴候がない1-2/dayと鎮痛のために、必要に応じて鎮痛剤投与</li></ol> IV。包帯除去（6-7日後に移植） <ol><li>承認された手順を使用してマウスを麻酔</li><li>絹の縫合糸を切り取り、削除する</li><li>慎重に包帯（移植片を妨害しないようにして作る）を切って、ゆっくりと除去、平滑末端のはさみの使い方</li><li>清潔なケージにマウスを返す</li><li>痂皮形成や収縮の徴候のための包帯除去後に移植片〜8時間を確認してください。存在する場合、移植片は、血管が発達するために失敗した可能性があると技術的障害を考慮する必要があります。 </li><li>拒絶反応の兆候を毎日監視する（通常ドナー組織の〜80％壊死として定義） </li><li>同種皮膚移植は、通常、D8 - 12術後の間に拒否されます。 </li></ol> V.役立つヒント<ol><li>耳の付け根にドナーの組織の近くに収穫することになります：1）移植片のサイズを最大化、および2）の分割を容易にする均一な腹側オーバーハングを提供</li><li>常に受信者を剃るための新たなカミソリの刃を使用してください</li><li>鎮痛剤は、IPを与えられている場合は、（移植片を破壊する可能性を最小限に抑える）術後の処理を最小限に抑えるための手順を移植する前に管理</li><li>可能であれば、包帯を適用する前に手袋を外す。手袋は、包帯を固執し、アプリケーションを複雑にする傾向がある</li><li>包帯最後に、単一の縫合糸を配置することで早期の除去を最小限に抑えることができます</li><li>タイトとして許可されているように包帯をラップし、縫合糸を配置する前に呼吸を評価する。包帯がきつすぎる場合、動物が呼吸を停止します。包帯が緩すぎる場合は、動物はそれを削除します。 </li></ol>

The authors have nothing to disclose.

このビデオの記事で示すように、皮膚の移植モデルでは、同種T細胞応答を監視するための迅速かつ容易な方法です。同種皮膚移植は、アプリケーションの広い範囲を持って、アッセイするために免疫抑制剤と寛容の誘導さまざまなプロトコルの効率性を利用されている、だけでなく、様々なブロッキング抗体の生物学的効果を調査する。

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<td>Micro-dissecting forceps (curved)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4142</td>
<td>&nbsp;</td>
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<td>Rat-tooth forceps (1x2 teeth, straight)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11053-10</td>
<td>n=2</td>
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<td>Strabismus scissors (blunt end)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14074-11</td>
<td>&nbsp;</td>
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<td>Iris forceps (curved)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11065-07</td>
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<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15006-09</td>
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<td>Halsey micro needle holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12500-12</td>
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<td>430167</td>
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<td>Monoject</td>
<td>8881 600145</td>
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<td>Solon</td>
<td>362</td>
<td>&nbsp;</td>
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<td>Single edge razor blades (#9)</td>
<td>&nbsp;</td>
<td>Smith</td>
<td>67-0238</td>
<td>&nbsp;</td>
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<td>Silk suture (2-0; PS-2)</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>583H</td>
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murine skin transplantation

最も厳しいと、少なくとも技術的に困難なモデルの一つとして、皮膚移植は、MHC -異種ドナー抗原に対するアッセイ宿主T細胞応答の標準的な方法です。このビデオ記事の目的は、マウスモデルを用いた皮膚移植のステップバイステップの視覚的なデモンストレーションで視聴者を提供することです。 2）移植のために受信者を準備する、3）皮膚移植、4）包帯の除去および監視の移植片拒絶反応、5）役立つヒント1）収穫ドナーの皮膚：プロトコルは、5つの主要なコンポーネントに分割されています。一度堪能、手順自体は、実行するために&lt;10分を取る必要があります。

Watch this Scientific Journal Video about Murine Skin Transplantation at JoVE.com

Murine Skin Transplantation

同種皮膚移植は、MHC -異種ドナー抗原に対するアッセイ宿主T細胞応答の標準モデルです。このビデオ記事はBALB/c--&gt; C57BL / 6皮膚移植を行うことに関係する各ステップの視覚的なチュートリアルが用意されています。

マウス皮膚移植

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Research

JoVE Journal

Biology

22.4K ビュー.  University of California, Irvine (UCI). 同種皮膚移植は、MHC -異種ドナー抗原に対するアッセイ宿主T細胞応答の標準モデルです。このビデオ記事はBALB/c--> C57BL / 6皮膚移植を行うことに関係する各ステップの視覚的なチュートリアルが用意されています。

ビデオ: Murine Skin Transplantation

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

マウスにおける異所心臓移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

生物学

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

マウスにおける小腸移植

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

マウス腎移植の手順

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

ヒトCD40活性化B細胞の生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植によるキメラゼブラフィッシュ胚の生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

マウスでXenografting胚細胞移植と精巣組織

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

プラナリアの固定化、部分照射し、組織移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

全細胞集団の単一細胞レベルでサイクリンBの分解を勉強

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

筋衛星細胞の単離、培養、および移植

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

バレット食道の細胞の特徴のための免疫蛍光法

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...