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1。準備<ol><li>培養皿の準備<ol><li> 4で解凍コラーゲンタイプIV O / N°C、0.1％滅菌酢酸に溶かし。使用するボリュームは、細胞が播種されるようにされている料理の種類や数によって異なります。 2μgの個/ cm 2を使用します。 </li><li>室温で1時間、少なくとも料理をインキュベートし、蒸留水（A. tridest）で2回洗浄する。 </li><li>料理は適切に乾燥させます。 </li></ol></li><li>媒体の補充<ol><li> 、500mlの培地に1.75グラムのブドウ糖を追加することで、最大4.5グラム/ Lに血糖値を上げます。培地600ミリオスモルへを調整するには、それぞれ、200ミリオスモルと100ミリオスモルによって浸透圧を高めるために、100mMのNaClと100mMの尿素を加える。 </li><li> 600ミリオスモルDMEM培地に1％非必須アミノ酸、1％ultroser、500μMの非添加、20 U / mlのナイスタチン及び0.25μgの/ mlのゲンタマイシン（50μgの/ mlの濃度とストック溶液の1:200希釈）を追加新たに準備の日に。 </li></ol></李&gt; <li>酵素溶液<ol><li> DPBS含有0.25μgの/ mlのゲンタマイシンおよびナイスタチンに1 mg / mlのヒアルロニダーゼおよび2.2 mg / mlのコラゲナーゼを追加します。 2腎臓内部の髄質の消化のため、この酵素溶液1mlを準備します。溶液を濾過滅菌しなければならず、50ミリリットルファルコンチューブに格納されている。 </li></ol></li></ol> 2。ダクト（IMCD）細胞を回収初代ラットインナー髄の栽培すべての動物の取り扱い手順は、ローカルおよび連邦法に準拠して行わなければならない。 <ol><li> CO 2のラット（CO 2で飽和ボックスで）またはイソフルラン（; 2から2.5パーセントイソフルランとエアーフロー350〜400ミリリットル/分のU-400麻酔ユニット、Univentor株式会社、マルタ）Anaesthetize。白っぽい内側と外側の暗い髄質の境界の正確な検出を可能出血のために動物の首を刈る。また、出血が孤立し、赤血球の数を最小限に抑えることができます。腎臓を取り出し、氷の上で（上記参照）ゲンタマイシンおよびナイスタチンを補っ滅菌DPBSでそれらを洗ってください。すべてのさらなるステップは、滅菌クリーンベンチの条件下で実施されるべきである。 </li><li>過剰な液体を除去する滅菌湿布にDPBS溶液から腎臓を転送します。滅菌ピンセットとハサミで脂肪腎臓カプセルを取り外します。 </li><li>腎臓はまだ圧縮で固定された、外側（皮質）側の縦軸横に少しカット。完全にカットスルーするのが、腎盂に接続し、一枚でそれを残していないことを確認してください。 </li><li>湾曲したハサミで丁寧にロゼきらめくアウター髄質に囲まれている白っぽい内側の髄質を解剖。氷の上を60mm培養皿にゲンタマイシンおよびナイスタチンを含む、DPBSでそれらを収集します。 </li><li>すべて髄質たら氷の上に分離し、収集され、それらは単にバッファで覆われているレベルにDPBSの量を減らす。 5のキューブに髄質をカットする滅菌カミソリの刃を使用して、ミリメートル3。 </li><li>炎に簡単にそれを保持することによって溶融、滅菌パスツールピペットの鋭い端を丸める。ピペットは、続行する前に冷却されていることを確認します。 </li><li>丸みを帯びたパスツールピペットを用いて、ヒアルロニダーゼおよびコラゲナーゼ（1.3.1を参照）を用いて新たに調製滅菌酵素溶液を含有する50ミリリットルファルコンチューブに組織懸濁液を移す。しっかりと蓋を閉め、37℃で1.5のための定期的な卓上水浴中で連続攪拌（220回転）の下でインキュベート - 2時間。 </li><li>サスペンションを均一混濁になるまで、懸濁液の粉砕のために、ラウンドパスツールピペット（上記参照）と、上下に数回混合物をピペット。 </li><li> 300 xgで5分間16℃の懸濁液を遠心上清を捨て、再びゲンタマイシンおよびナイスタチンと遠心分離機を含む、DPBSで徹底的にペレットを再懸濁します。一度この洗浄工程を繰り返します。 </li><li>完全培地と種子、それらをCで細胞を再懸濁しulture料理および/またはマイクロタイターウェルプレート。 2髄質（1動物）の調製は、1を60mm培養皿に十分な細胞が得られます。 </li><li> 24時間後、600ミリオスモルDMEMで細胞を2回洗浄し、新鮮な培地を追加します。来週中に、細胞を2〜3回（ 図1）を洗浄する必要があります。 6-8日播種後、IMCD細胞を実験に使用することができる。実験を開始する前に、24時間非添加およびナイスタチンを含まない培地でそれらをインキュベートすることを忘れないでください。そうでなければAQP2は形質膜に排他的に配置される。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

私たちは、初代ラットIMCD細胞の調製と培養するための詳細なプロトコルを提示します。アプローチでは、1匹のラット20から細胞21 cm 2とまで得られます。実験は、標準的な細胞培養装置を必要とし、約6時間以内に一人で行うことができる。したがって、このアプローチは、標準的な実験方法として好適である。 初代ラットIMCD細胞を96ウェルプレートから6...

A correction was made to <a href="http://www.jove.com/video/50366">Culturing Primary Rat Inner Medullary Collecting Duct Cells</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Enno Klussmann instead of: Enno Klussman

A correction to author list has been made for article Culturing Primary Rat Inner Medullary Collecting Duct Cells.

Erratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells

腎集合管主な細胞では、アルギニン·バソプレシン（AVP）は、原形質膜への水チャネルアクアポリン2（AQP2）の挿入を刺激による水再吸収を制御します。 AVPは、アデニリルシクラーゼことにより、cAMP形成を刺激するGタンパク質共役型バソプレッシン-2受容体（V2R）に結合する。このシグナリングカスケードの開始は、プロテインキナーゼA（PKA）の活性化をもたらす。 PKAは、原形質膜への細胞内小胞からの再分配のための重要なトリガーである、256（S256）セリンでAQP2をリン酸化する。膜挿入は、浸透勾配に沿って水の再吸収と微調整の体内の水分の恒常性を容易にします。 原因AVP分泌またはAVP-活性化シグナリング原因かの収差AVP媒介水再吸収の調節不全は、深刻な病気に関連付けられています。 V2RまたはAQP2の突然変異によって引き起こさ減少水の再吸収はELEVのに対し、腎性尿崩症につながるated血漿AVPレベルは、慢性心不全および不適切な抗利尿ホルモンの分泌（SIADH）の症候群などの心血管疾患の過度の水再吸収に関連付けられています。 AVP媒介水再吸収の重要性だけではなく、病気でその意義に由来する。 AQP2有利子に小胞と形質膜と融合のAVP-誘発転座は、現在十分に理解されていない厳密にcAMP依存性エキソサイトーシス過程を表します。 cAMP依存性エキソサイトーシスのための他の例としては、腎臓とHにおけるレニン分泌+胃の中に分泌されています。したがって、腎主要細胞内AQP2-転座を支配する分子メカニズムの解明だけでなく、他の細胞型で同様の分子プロセスを理解するのに役立つだけでなく、AVP-媒介水再吸収の障害に関連する疾患の治療のための新しい治療法に道を開くことが。 EAQP2を制御lucidatingメカニズムは、集合管主な細胞モデルを必要とします。このため、異なる哺乳類の腎臓細胞株の数が用意されています。しかしながら、これらのモデルはいくつかの欠点を有する。多くの細胞系におけるAQP2のタンパク質レベルが低い（ 表1; MDCK M1、HEK293、COS-7、、LLC-PK1）1-10、異所性に過剰発現する他の人間（MCD4、WT10）11,12またはラット（CD8） 13 AQP2。 MCD4およびCOS-7細胞ではV2受容体が発現していない。我々の知る限りそこ腎髄質内層集合管、AQP2式が最も豊富である腎臓のその部分から派生して利用できるモデルなど恒久的な細胞株はありませんが、皮質集合管1,11,13-16から。このような細胞モデルは、広く使われている例不死mpkCCD（ 例えば 17）、または最近設立mTERT-CCD 14細胞株のためのものです。両方の細胞株内因的に発現AQP2とV2Rが、それらは由来しているので皮質集合ダクトは、最も可能性の高いダクト（IMCD）細胞を収集し、内側延髄に比べ異なるプロテオームが含まれています。彼らは、例えば別のNaを表現しなければならない+交通システム。 ここでは、V2RとAQP2内因を表現文化プライマリーIMCD細胞にプロトコルを提示します。このモデルは、したがって、最も近い腎集ダクト内の生理学的状況を示す。文化を確立したように、標準実験装置他の研究室は、簡単にこのアプローチを採用することができます必要があります。

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			<td>Company Catalog</td>
			<td>Number</td>
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			<td>Collagen Type IV Mouse</td>
			<td>BD Biosciences</td>
			<td>356233</td>
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			<td>Hyaluronidase</td>
			<td>SIGMA</td>
			<td>H6254</td>
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			<td>Collagenase type CLS-II</td>
			<td>Biochrom AG</td>
			<td>C2-22</td>
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			<td>DMEM + GlutMAX</td>
			<td>Invitrogen GIBCO</td>
			<td>21885108</td>
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			<td>Nystatin</td>
			<td>SIGMA</td>
			<td>N4014</td>
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			<td>Ultroser G</td>
			<td>Cytogen</td>
			<td>15950-017</td>
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			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
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			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
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			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
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culturing primary rat inner medullary collecting duct cells

アルギニン·バソプレシン（AVP）はダクト校長細胞とそれによって微調整の体水分ホメオスタシスを集めることにより、水腎再吸収を促進します。 AVPは、細胞の表面上バソプレシンV2受容体（V2R）に結合し、それによって、cAMPの合成を誘導する。これは、水路アクアポリン2（AQP2）のリン酸化の変化をもたらす細胞内シグナル伝達プロセスを刺激する。プロテインキナーゼは、AQP2をphoshorylates、それによって主要な尿からの水の再吸収を促進する形質膜に細胞内の小胞からAQP2の転座をトリガします。主要細胞内の下垂体またはAVP-活性化シグナリングからAVP放出の収差はそれぞれ、中央または腎性尿崩症を引き起こす可能性があり、血中血漿AVPレベルは、慢性心不全および抗利尿ホルモン不適合分泌症候群などの心血管疾患に関連付けられています。 ここでは、cultivati​​ためのプロトコルを提示する急行V2RとAQP2内因ダクト（IMCD）細胞を収集初代ラット内側の延髄の上。細胞は、AQP2の制御の根底にある分子メカニズムを解明するのに適しているので、AVP媒介性の水再吸収の調節不全に関連する疾患の治療のための新規薬剤標的を発見する。 IMCDネズミ腎細胞は、内側髄質から得られ、六から八日播種後の実験のために使用される。 IMCD細胞は規則的な細胞培養ディッシュ、フラスコ、異なる形式のマイクロタイタープレートで培養することができ、手順は数時間を必要とし、標準的な細胞培養実験室に適している。

初代ラットIMCD細胞の正常な栽培は6-8日播種後コンフルエントな単層（ 図2）になります。 60ミリメートル培養皿あたり約6×10 6個の細胞があります。これらはIV型コラーゲン、基底膜成分を18でコーティングしたような細胞がしっかりと、培養皿に付着する。したがって、IMCD細胞はいくつかの徹底した洗浄手順の中でも切り離すことはありません。最大培養細胞の8...

Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Culturing Primary Rat Inner Medullary Collecting Duct Cells

アルギニン·バソプレシン（AVP）は腎校長細胞による水の再吸収を促進を通じて体内の水分の恒常性の微調整を制御します。ここでは、AVP-媒介水再吸収の根底にある分子機構の解明に適した管細胞を集め初代ラット内側の延髄の栽培のためのプロトコルを提示します。

管細胞の収集培養初代ラットインナー髄

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

culturing-primary-rat-inner-medullary-collecting-duct-cells

Research

JoVE Journal

Biology

14.7K ビュー.  Max-Delbrück-Center for Molecular Medicine. アルギニン·バソプレシン（AVP）は腎校長細胞による水の再吸収を促進を通じて体内の水分の恒常性の微調整を制御します。ここでは、AVP-媒介水再吸収の根底にある分子機構の解明に適した管細胞を集め初代ラット内側の延髄の栽培のためのプロトコルを提示します。

ビデオ: Culturing Primary Rat Inner Medullary Collecting Duct Cells

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

ラット上頚神経節（SCG）から培養交感神経のためのプロトコル

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

生物学

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成体ラットの心筋細胞の初代培養

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

外植片から成長した初代ヒト気管支上皮細胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

マウス気管から基底細胞および粘膜下腺管細胞の単離

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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 ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

エア·液界面での人間の鼻上皮細胞の培養

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

培養胚のための方法<em&gt; C.虫</em&gt;細胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

初代培養ラット副腎皮質細胞とステロイド ホルモン産生機能の試金

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...