JLNは、MH 68347によってサポートされていました。

海馬分離に先立って海馬の分離を開始する前に、すべてのツールが無菌であることを確認してください。 70％エタノールで文化のフードを下にスプレーし、及びフード内のツールを配置します。と10cmのシャーレ、滅菌ポリ- Lコーティングされたガラス製カバースリップ、ピペッターやヒント、使い捨てピペット、および電動ピペッター - あなたは6が必要になります。この時点から、適切な無菌操作を使用することを忘れないでください。 <ol><li>水浴の電源を入れ、それを37℃にまで加熱されていることを確認してください</li><li> 4℃で保存されている以下のソリューション° Cが必要になります。 <ul><li>改変イーグル培地（MEM） </li><li> Neurobasal </li><li>ハンクス緩衝生理食塩溶液（HBSS） </li><li>ホウ酸塩緩衝液</li><li>ピルビン酸ナトリウム液</li><li>蒸留水で滅菌濾過20％グルコース溶液 </li><li>フードにそれらを配置する前に、70％エタノールですべてのボトルを下に散布することを確認してください。 </li></ul></li><li>冷凍庫から、これらの冷凍のソリューションを取ると温水浴中にそれらを配置します。 <ul><li>ウマ血清、5 mLを</li><li> B - 27サプリメントを1mlのアリコート</li><li> 100X抗生物質を1ml​​のアリコート（ペニシリンプラスストレプトマイシン） </li><li> L -グルタミンのと0.5mlのアリコート</li></ul></li><li>ソリューションが融解した後、水浴からそれらを取り出して70％エタノールでそれらを吹きかけ、そしてフードの中に置いてください。今すぐメッキとNeurobasal培地を調製することができる。 </li><li>ソリューションの準備が完了した後、海馬の分離を行いながらウォームアップするためにしっかりと容器にフタをし、37℃の槽の中に置いてください。 </li><li>また、冷凍庫の外プロテアーゼトリプシン（2.5％）のアリコートを取り、水浴に入れてください。トリプシンは、次のステップで分離されて解剖海馬を、ダイジェストになります。 </li><li> 、15ミリリットルコニカルチューブを取るHBSSでそれを入力し、治療群とそれにラベルを付ける。それは、孤立した海馬は、次のステップで収集されることをここにある。 </li></ol>海馬アイソレーション<ol><li>海馬の分離を開始するには、新生児の子犬が汚れていないと、そのミルクのバンド、胎盤、およびその母親によって削除臍帯を持っていることを確認してください。 </li><li>きれいに、そしてボンネットに配置する70％エタノールで、ペトリ皿にスプレーして子犬を置きます。動物は、培養前に即座に安楽死させる。 </li><li>さらに殺菌のために一皿から子犬を取る、70％エタノールに子犬を浸し、次に滅菌HBSS中で2回洗浄に。 </li><li>はさみで本体からヘッドを取り外します。同じハサミを使って、皮膚や頭蓋骨を介してカット。 </li><li>離れて脳から頭蓋骨をはがし、細かいピンセットを使用し、滅菌HBSSを少量含まれている小型のシャーレに脳を置く。 </li><li>大脳半球バックピール。海馬は、内側側頭葉にある小さな、タツノオトシゴの形をした構造です。 </li><li> 15 mlのチューブにHBSS 3mlに海馬と場所を削除します。各子犬にこれらのステップを繰り返し、15 mlチューブにそれぞれの分離された海馬を置きます。今、それは単一の細胞に組織を解離する時間です。 </li></ol>海馬細胞解離<ol><li>すべての海馬が分離された後、HBSSで4.5 mlに15 mlのチューブを埋める。 </li><li>水浴、エタノールでスプレー、そしてフードの場所からトリプシンを削除します。チューブにトリプシンの0.5 mlを加え、そして37℃で15分間反応させます℃に</li><li>フードでは、チューブの底に沈殿している海馬を乱さないように注意しながら、滅菌ピペットでチューブからHBSS /トリプシン溶液を除去。優しくチューブと渦巻きにHBSSを5mlを追加。 ℃で5分間37℃インキュベートする。 </li><li>古いHBSSを除去し、新鮮な溶液と置き換えて、二度この手順を繰り返します。 </li><li>私は冷凍庫からDNアーゼのアリコートを取り出してください。 4.5ミリリットルのHBSSで海馬にDNaseの0.5 mlを追加します。 DNアーゼ、酵素不活性化を促進するために追加されます。 </li><li>ソリューションをひいて粉にする（または、上下にピペッティング）が均一になるまで。ホモジネートに気泡を導入しないように注意してください。 </li><li>トリパンブルー排除法で細胞の生存を決定する。 </li><li> 6から8までカバースリップを含む10cmペトリ皿を取る、とメッキの培地10mlを追加してください。カバースリップを含む皿に希望のセル数をピペ​​ット。細胞を分散し、カバースリップが重複していないことを確認して静かに渦巻く。 </li><li>細胞は5％CO 2で加湿37℃インキュベーターで2〜4時間のために添付することができます。細胞は生存可能であり、接続されていることを確認した後、Neurobasal培地を含む個々の皿にカバースリップを転送する。インキュベーターに戻し、これらの皿を置きます。 </li><li>週に一度、必要に応じて、新鮮なNeurobasal培地と実験的な治療で培地の3分の1を交換してください。今、培養中のこれらの細胞の機能的特性についても研究する準備が整いました。 </li></ol>海馬ニューロンのフラ-2カルシウムイメージング<ol><li>培養海馬神経細胞が48時間（ただし、それ以前）のためにin vitroでされた後、カルシウムのイメージング実験を開始することができます。この時点で、これらのニューロンは、プロセスを拡張し始めているはず。カルシウムイメージングに最適な年齢範囲は 、in vitro 3日〜7です。 </li><li>フラ-2と文化をロードすると、37℃で30分間午前℃で、それらがどのサンプルカルシウム指示薬染料および512ビットのCCDカメラを励起するキセノンアークランプを使用して20倍対物レンズ、すべての150msの下でイメージングすることができる後。 </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells, 2nd Edition. , (1998).</li></ol>

このプロトコルは、ゲイリーバンカーと金ゴズリン1の精液の仕事の修正として開発されました。この本は、培養細胞に興味を持つ人にとって不可欠な資源です - だけでなく、ニューロンに富んだ文化が現在のプロトコルで説明しています。 細胞培養における三大重要な要素は、次のとおりです。不妊、速度、使用する培地の選択。 無菌性 -層流/無菌フード、無菌条件、およ...

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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Antibiotic antimitotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240062</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504044</td>
<td>Serum free</td>
</tr>
<tr>
<td>Boric Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B6768</td>
<td>&nbsp;</td>
<tr>
<td>Dnase I</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>DN25</td>
<td>&nbsp;</td>
<tr>
<td>Fura-2, AM cell permeant</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>F1221</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14185052</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630080</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>51200038</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12348017</td>
<td>Without phenol red</td>
</tr>
<tr>
<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6282</td>
<td>&nbsp;</td>
<tr>
<td>Pyruvic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Tetraborate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 2.5% (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15090046</td>
<td>&nbsp;</td>		</tbody>
		</table>
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primary culture of hippocampal neurons from p0 newborn rats

海馬ニューロンの生理学的特性は、一般的には、特に理由の学習と記憶における海馬の関与が、研究されています。主海馬細胞の培養は、神経科学者は、個々の細胞と単シナプスレベルでのニューロンの活性および特性を調べることができます。このビデオでは、我々は、新生児ラットから主海馬細胞を分離し、成長する方法を紹介します。海馬は2〜3分ほどの短い内の各新生児の動物から単離することが、文化は最大2週間のために維持することができます。我々はまた、簡単にレシオメトリックカルシウムイメージングのためのこれらの海馬神経細胞を使用する方法について説明します。このプロトコルは、修正なしに少しと、海馬のためのプロセスを説明しながら、それは脳の他の地域に適用することができます。

Watch this Scientific Journal Video about Primary Culture of Hippocampal Neurons from P0 Newborn Rats at JoVE.com

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

解剖や個々の脳領域からの細胞の増殖は、細胞と生理的パラメータの調査を容易にします。我々は、無血清環境でのニューロンに富んだ文化を生産する主要な細胞培養のための方法を説明します。

P0新生児ラットからの海馬ニューロンの初代培養

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

primary-culture-of-hippocampal-neurons-from-p0-newborn-rats

Research

JoVE Journal

Biology

76.5K ビュー.  Michigan State University (MSU). 解剖や個々の脳領域からの細胞の増殖は、細胞と生理的パラメータの調査を容易にします。我々は、無血清環境でのニューロンに富んだ文化を生産する主要な細胞培養のための方法を説明します。

ビデオ: Primary Culture of Hippocampal Neurons from P0 Newborn Rats

Gene-gun Transfection of Hippocampal Neurons

Hi, I'm Kma. I work in Case Lab at Brown University Clean Slicer stage with 70%ethanol. Place plate insert into six.Well plate with medium and incubate at 37 degree whole brain with tweezers between midbrain and four brainin. Isolate hippocampus on one side, isolate hippocampus from the second side. Place hippocampi onto A PBS drop.Aspirate out the PBS make sagittal sections using the tissue slicer. Repeat transfer slicers onto a perish by squatting with PBS wash sections by adding and aspirating PBS and medium gently. Separate sections with tweezers.Wash sections with PBS and media. Again, transfer sections onto the plate and insert. Aspirate out the media in sections using tweezers.Attach gene gun hose to the helium cylinder. Attach barrel to gene gun. Fill the magazine with bullets.Insert magazine into the Jing gun. Adjust the pressure on the helium tank shoot. EGFP coated beads onto the sections.And now.

海馬ニューロンの遺伝子郡のトランスフェクション

生物学

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

初代培養海馬ニューロンにおける高密度コア小胞のライブイメージング

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

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 ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

成体マウスから海馬歯状回の解剖

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

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 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 Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

ミニチュアブタからの原発性大動脈内皮細胞の単離と培養

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

新生児マウスからの白色および褐色の前読細胞の分離と分化

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

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 ...