この作品は、JYに健康グラント5KO8DK085136-02の国立研究所によってサポートされていました。

1。結腸組織を得るマウス研究の根拠と目的を詳細に動物実験を行うためのプロトコルでは、提出され動物実験的監督の私たちの事業オフィスによって承認された。 <ol><li>頸椎脱臼に続いて吸入イソフルランでマウスを安楽死させる。 </li><li>腹側正中切開を行い、離れて小腸を撤回し、コロンを識別します。横方向に膀胱と子宮（存在する場合）を撤回し、恥骨を特定し、直腸を露出させ細かいハサミでカット。 </li><li>大腸から直腸の背側腸間膜を解剖し、肛門から盲腸までコロンを動員し、小腸からの結腸を切断し、それを横断する。 </li><li>氷冷リン酸緩衝生理食塩水（PBS）中に試料を置く。 </li><li>氷冷PBSで10ミリリットルの注射器を記入し、コロンをフラッシュし、大腸OPEを切断し、その内容をクリアするために数回ルーメンN長さ方向はさみを使って。 </li><li>氷冷PBSの20ミリリットルを含む50ミリリットルコニカルチューブにコロンを置きます。結腸は、任意の特定の方向に配置される必要はない。流体が無い粒子状物質と透明になるまで、PBSを交換して、コロンを洗ってください。 </li></ol>人間この組織だけでなく、潜在的なリスクとベネフィットの評価を得ることの重要性を詳述手術患者からのヒト組織を得るためのプロトコルは、ヒューマン研究保護プログラムの我々の事業オフィスから提出され、承認されました。 <ol><li>大腸外科医との共同研究は、研究のためのヒト組織を得ることができることが必要である。ヒトの大腸の組織を得るためのプロトコルが提出し、治験審査委員会によって承認されなければならない。 </li><li>書面によるインフォームドコンセントを、外科手術の前に患者から得られる。 </li><li>外科医は、スタンドに結腸切除を行いますARDファッション。結腸は、患者から除去された後、試料を直ちに病理に取り込まれる。病理学的評価のために必要ではない結腸壁の全厚部0.5が設けられており、すぐに氷冷したPBS上に配置される。この標本は、大腸間膜を含めるべきではなく、脂肪の存在は消化過程に影響を与えますので、大網付属は、同様に削除する必要があります。 </li></ol> 2。上皮細胞はぎ取る<ol><li> 50ミリリットルコニカルチューブ内のHBSS中の5ミリモルのEDTA（室温）25mlに、全マウス結腸をインキュベートする。 15分間空気浴（250回転）しながら37℃にコニカルチューブを配置します。 15分後、慎重に液体を捨て、5のEDTA 25mlでチューブを補充、5回の洗浄の合計のため繰り返します。ヒトの大腸では、病理医から結腸壁の2分​​の1インチのストリップを得た後、4つの等しいサイズのピースにハサミでコロンをカット。 1 50ミリリットルコニカルチューブにピースの2を置き、独立した50ミリリットルコニカルチューブに結腸組織の残りの2個を配置。そして、前述したように、HBSS中5mMのEDTA 25mlでインキュベートおよび洗浄を行う。 </li><li>氷冷PBS 20mlに二度、コロンを洗浄します。 </li><li> RPMI-5の20ミリリットル、ディスパーゼの10、U、およびコラゲナーゼDの2000 U（室温）を含む新しい50ミリリットルコニカルチューブにマウス結腸組織を置きます。ヒト大腸組織のために、ディスパーゼおよびコラゲナーゼ（20 U字ディスパーゼ、4000のUコラゲナーゼD）の二倍の量を使用しています。 </li><li> （水平に置いた場合は、あまりにも多くの通気や細胞の損傷がある）、垂直に配向37℃の揺れの空気浴中のチューブを配置します。最大60分間、250rpmで振とうする。ディスパーゼおよびコラゲナーゼDに暴露した後、結腸組織を消化するために開始され、結腸組織は、糸を見て開始されます。これは通常約30分かかります。コロンは、この外観を有する場合には、酵素からコロンを削除します。 </li><li>組織を分割するには、上下2〜4回、各チューブを横に振る。 P5分間、卓上遠心機（4℃）で200×gで組織をエレト。慎重に上清を捨て、捨てます。 </li></ol> 3。筋線維芽細胞の単離および培養<ol><li>を10mlのACK溶解緩衝液（47°C）を添加することにより、各ペレットを再懸濁。遠心分離器は、再び5分（4℃）のための200×gで、捨て、上澄みを捨てる。 </li><li>ピペットを用いて10ミリリットルのRPMI-5を追加して、各ペレットを再懸濁します。 </li><li> 100ミリメートルのTC処理皿に10ミリリットルピペットを用いて70ミクロンメッシュストレーナーを通して細胞懸濁液（5ミリリットル）の半分を渡す。その後RPMI-5の追加の15ミリリットルを追加します。別の100ミリメートルのTC処理皿に細胞懸濁液の残りの5ミリリットルを繰り返します。一つのマウス大腸は、したがって、2 100ミリメートルのTC処理の料理が得られます。ヒトの大腸組織の一つ½インチのストリップは4 100 mmのTC処理の料理の合計が得られます。 </li><li> 37℃、10％CO 2インキュベーター内で組織培養皿を置き培養条件は同じfはあるいは、マウスおよびヒト初代細胞。 </li><li> 3時間後、そっとのHBSS 2回交換して非接着細胞を洗い流してください。破片の浮動を静かに洗浄する必要があります。接着細胞は、上皮細胞、マクロファージおよび筋線維芽細胞で構成されている。一週間播種後、唯一の除算を筋線維芽細胞、マクロファージおよび上皮細胞は最初の継代後に老化。新鮮なRPMI-5を添加し、細胞培養物中の細胞を成長させる。 </li><li>細胞を5分間トリプシン処理により継代すると2:1に分割されています。 </li></ol>略語 HBSS：ハンクス液、RPMI-5：ロズウェルパーク記念研究所媒体、ACK：塩化アンモニウムカリウム、FCS：ウシ胎児血清、TC処理：組織培養処理ソリューション ACK溶解緩衝液- （4.15グラムNH 4 Clで0.5グラムKHCO 3、18.6ミリグラムのNa 2 EDTAを400 mlのH 2 O、1 N HClでpH7.2〜7.4にpHを調整する）。フィルタsterilizEでの0.2μmのフィルターや店舗を通じて4℃のRPMI-5 - （500ミリリットルにする方法：454.5ミリリットルのRPMI、25ミリリットルのFCS、5ミリリットル200 mM L-グルタミン、5ミリリットルの1M HEPES、pH7.4で、5ミリリットルを100 mMピルビン酸ナトリウムを5ml 100×ペニシリン - ストレプトマイシン、PBS 500 mlの50 mMのB-ME）。

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著者らは、開示することは何もありません。

マウスまたはヒトの結腸のいずれかを使用するときの一般的な手法は同様である。この技術はまた、小腸からの筋線維芽細胞を単離するために使用することができる。 1からの筋線維芽細胞を単離するための具体的な詳細。マウスと2。ヒトの大腸を以下に説明します。 1。マウス結腸マウス結腸の灌注は、結腸の一端に16 Gポッパー平滑末端針4を取り付...

胃腸（GI）管の機能の調節は、粘膜層とその下にある間葉との間の複雑で動的な相互作用を伴う。これらの相互作用は、通常は恒常性を維持するのに役立つだけでなく、病的条件1での疾患状態の発症につながる可能性がある。筋線維芽細胞は、粘膜の再生、修復、および線維症2を含む重大な胃腸管プロセスの数を調節する細胞亜集団を包囲してパラクリン様式で通信する上皮層への直下に位置する間質細胞の亜集団である。 18 CO細胞株は、それがインサイチュ筋線維芽細胞の一次の特徴の多くを共有しているため、広く筋線維芽細胞の機能を研究するために使用されているヒト結腸筋線維芽細胞株の一例である。これらは、タンパク質-平滑筋アクチン（α-SMA）の発現およびビメンチン、並びに多数の発現を含む例えば、上皮成長因子受容体、又はリゾホスファチジン酸、3,4に対する受容体などの細胞表面受容体。なぜならこれらの共有超微細構造特性、ならびに生物学的機能5の類似性、18 CO細胞株は、炎症性腸疾患または結腸直腸癌3,6のような多くの疾患状態のコンテキストで筋線維芽細胞の機能を研究するために広く使用されている。しかし、細胞株を用いて固有の限界や懸念がある。これらは他の細胞集団との成長率との相互作用など、細胞の表現型および生物学的機能を変化させる、一次細胞から細胞株を区別し、経時的遺伝子型不安定性が含まれる。細胞株もまた、その使用に対する主な制限である消化管の微小環境（上皮、間質、および血管成分）を、通常の構成要素を欠いている。したがって、実験的な細胞株の研究から引き出された結論は、里を減少させるためにさらなる検証が必要で誤解のSK。 一次筋線維芽細胞は、細胞株7において同定実験結果を確認するために、ヒトおよびマウスの結腸組織から得ることができる。この方法は、もともとMahida ら 8によって記述され、我々のプロトコルは、マウスおよびヒト結腸9から一次筋線維芽細胞を単離するために用いることができる多くのよく説明される技術の一つであるした。結腸疾患の任意のマウスモデルについては、この技術は、GI 10,11を処理し、それらが隣接細胞とどのように対話するかを研究するために、一次筋線維芽細胞を単離するか、正常または病的両方に寄与するために使用することができる。この技術は、筋線維芽細胞は、治癒、線維症、および結腸直腸腺腫および癌腫の発生を粘膜に寄与する方法を研究するために使用することができる。一次筋線維芽細胞はまた、良性（炎症性腸疾患、狭窄、憩室炎）または悪性のc運転時に切除されたヒト結腸組織から得ることができるonditions。ヒトおよびマウスの結腸組織から単離した初代細胞は、細胞培養で増殖させ、継代の数が限られにわたって利用することができる。

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Dispase I</td>
 <td>Invitrogen/GIBCO</td>
 <td>#17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>#1 088 858</td>
 <td></td>
 </tr>
 <tr>
 <td>4 in, 16 G Popper blunt-end needle</td>
 <td>Fisher</td>
 <td>#14-825-16K</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of primary myofibroblasts from mouse and human colon tissue

筋線維芽細胞は、多くのGI機能におけるその重要な役割のためにかなりの注目を集めている胃腸（GI）管の間質細胞である。いくつかの筋線維芽細胞株は、インビトロでこれらの細胞を研究するために市販されているが、実験的な細胞培養条件に曝露された細胞株からの研究結果は、作業の過度に還元主義的性質に起因する固有の限界を有する。一次筋線維芽細胞の使用は、細胞株で特定された実験結果を確認するという点で大きな利点を提供しています。動物モデルからの一次筋線維芽細胞の単離は、より厳密に検討されて疾患状態を模倣する条件下での筋線維芽細胞の研究を可能にする。細胞は基礎疾患を持つ患者から直接来ているので、ヒト結腸組織からの一次筋線維芽細胞の単離は、間違いなく、最も関連性の高い実験データを提供しています。我々はUであり得る、十分に確立された技術を説明マウスおよびヒト結腸組織の両方からの一次筋線維芽細胞を分離するためにtilized。これらの単離された細胞は、上皮下腸の筋線維芽細胞と一致してα-平滑筋アクチンとビメンチン陽性、およびデスミン陰性であることが特徴づけられている。一次筋線維芽細胞は、細胞培養で増殖させ、継代の数が限られにわたって実験目的のために使用することができる。

一旦単離される、初代ヒト筋線維芽細胞は、細胞培養で増殖させ（継代4まで）の通路の限られた数の上で使用することができる。これらの細胞は腸管上皮下筋線維芽細胞5,7と一致して、平滑筋アクチンとビメンチン陽性であると特徴づけ、およびデスミン陰性（ 図1A）されています。それらはまた、特徴的な星状形態（ 図1B）を有する。一次筋線維芽細胞は...

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Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

筋線維芽細胞は、正常および疾患状態の両方において重要な生理学的プロセスを調節、胃腸管の有力な間質細胞である。我々は、マウスおよびインビトロ実験のために利用することができるヒト結腸組織の両方からの一次筋線維芽細胞の単離を可能にする技術が記載されている。

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

isolation-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

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JoVE Journal

Biology

19.5K ビュー.  UCLA. 筋線維芽細胞は、正常および疾患状態の両方において重要な生理学的プロセスを調節、胃腸管の有力な間質細胞である。我々は、マウスおよびインビトロ実験のために利用することができるヒト結腸組織の両方からの一次筋線維芽細胞の単離を可能にする技術が記載されている。 

ビデオ: Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

人間の死後の脳組織から神経細胞核の単離

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

生物学

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

原発腫瘍から免疫細胞の分離

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

マウスおよびヒト食道から筋線維芽細胞の単離

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心臓線維芽細胞と筋線維芽細胞の分離と特徴付け

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

腸管内窩からのネズミ原発結上皮単層の生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

成人マウス口蓋からの一次口腔角化細胞の分離と培養

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...