JLN는 MH 68347에 의해 지원되었다.

hippocampal 분리하기 전에 hippocampal 고립을 시작하기 전에 모든 도구 멸균되어 있는지 확인합니다. 70 % 에탄올과 문화 후드를 스프레이하고, 후드 내부의 도구를 놓으십시오. , 멸균 폴리 - L 코팅 유리 coverslips, pipettors 및 팁, 일회용 pipettes, 그리고 전기 pipettor과 10 cm 배양 접시 - 당신이 6 필요합니다. 이 시점부터는 올바른 살균 기술을 사용해야합니다. <ol><li> 물 목욕 켜고 그것이 37 ° C.까지 가열되어 있는지 확인합니다 </li><li> 4 저장되어있는 다음과 같은 솔루션 ° C가 필요합니다 : <ul><li> 수정된 이글의 중간 (멤) </li><li> Neurobasal </li><li> 행크의 버퍼 생리 식염수 (HBSS) </li><li> Borate 버퍼 솔루션 </li><li> 나트륨 pyruvate 솔루션 </li><li> 증류수에 멸균 필터링 20 % 포도당 용액 </li><li> 후드에서 그들을 배치하기 전에 70 %의 에탄올과 함께 모든 병을을 스프레이하십시오. </li></ul></li><li> 냉동실에서 이러한 냉동 솔루션을 가지고 온수 물 목욕에서 그들을 장소 : <ul><li> 말 혈청 5 ML의 나누어지는 </li><li> B - 27 보충의 한 ML의 나누어지는 </li><li> 100X 항생제의 한 ML의 나누어지는 (페니실린 스트렙토 마이신 플러스) </li><li> L - 글루타민 및 0.5 ML의 나누어지는 </li></ul></li><li> 솔루션은 해동 후, 물 목욕 그들을 데리고 70 % 에탄올과 그들을 스프레이하고, 후드에서 그들을 놓으십시오. 이제 도금과 Neurobasal 매체 준비하실 수 있습니다. </li><li> 해결책이 준비 후 hippocampal 고립을 수행하는 동안 워밍업을 단단히 용기 뚜껑과 37 ° C 목욕에서 그들을 놓으십시오. </li><li> 또한, 냉장고 밖으로 프로 테아제 트립신 (2.5 %)의 나누어지는를 타고 물 욕조에 넣습니다. 트립신은 다음 단계에서 격리됩니다 해부하는 해마를 소화합니다. </li><li> , 15ml 원뿔 튜브를 타고 HBSS로 기입하고, 치료 그룹으로 분류. 그것은 고립된 hippocampi는 다음 단계에서 수집됩니다 여기에 있습니다. </li></ol> Hippocampal 절연 <ol><li> hippocampal 격리를 시작하려면 신생아 새끼는 깨끗해하고 우유 밴드, placentas, 그들의 어머니에 의해 제거 탯줄 코드를 적이 있는지 확인하십시오. </li><li> 청소하고, 후드에서 그들을 곳으로 70 %의 에탄올과 배양 접시에있는 스프레이 새끼를 놓습니다. 동물은 euthanized culturing 바로 앞에 있습니다. </li><li> 자세한 살균 한 접시에서 강아지를 타고 70 % 에탄올로 강아지를 찍어 다음 멸균 HBSS 두 씻는다로. </li><li> 가위로 시체에서 머리를 제거합니다. 같은 가위를 사용하여 피부와 두개골을 통해 했네요. </li><li> 멀리 두뇌에서 두개골 껍질, 괜찮아요 족집게 한 켤레를 사용하여 멸균 HBSS의 작은 금액을 포함하는 작은 페트리 접시에 머리를 놓으십시오. </li><li> 대뇌 반구 다시 필. 해마는 중간 측두엽에있는 작은 해마 모양의 구조입니다. </li><li> 15 ML 튜브의 HBSS 3 ML에 해마과 장소를 제거합니다. 각각의 강아지와 함께 이러한 단계를 반복하고, 15 ML 튜브에 각각 분리된 해마를 놓으십시오. 이제 하나의 세포로 조직을 떼어 놓다 시간이 있습니다. </li></ol> Hippocampal 세포 분리 <ol><li> 모든 hippocampi가 고립되어 후 HBSS와 4.5 ML로 15 ML 튜브를 입력하십시오. </li><li> 후드에있는 물 목욕, 에탄올과 스프레이, 장소에서 트립신을 제거합니다. 튜브에 트립신 0.5 ML를 추가하고 37 15 분 동안 품어 ° C. </li><li> 후드에서 튜브의 바닥에 정착 hippocampi을 방해하지 않도록주의하고, 멸균 피펫으로 튜브에서 HBSS / 트립신 용액을 제거합니다. 부드럽게 관과 소용돌이에 HBSS 5 ML을 추가합니다. ° C 5 분 37 알을 품다. </li><li> 이전 HBSS를 제거하고 신선한 솔루션으로 대체 두번이 단계를 반복합니다. </li><li> 냉동고의 I가 DNAse의 나누어지는 가져가라. 4.5 ML의 HBSS에 hippocampi에 DNase 0.5 ML를 추가합니다. DNAse는 효소 불활 성화를 촉진에 추가됩니다. </li><li> 솔루션을 씹다 (또는 그리고 아래로 피펫)는 동질 때까지. homogenate에 거품을 소개하지 않도록주의하십시오. </li><li> trypan 블루 제외 메서드를 사용하여 세포 생존을 확인합니다. </li><li> 6-8 coverslips을 포함 10cm 페트리 접시를 타고, 도금 매체 10 ML를 추가합니다. coverslips 들어있는 접시에 세포의 원하는 번호를 피펫. 소용돌이는 부드럽게 세포를 분산하고, coverslips가 중복되지 않도록합니다. </li><li> 세포 5 % CO 2 humidified 37 ° C 배양기에서 2-4시간 위해 첨부할 수 있습니다. 세포가 가능한되며 첨부 것을 확인한 후, Neurobasal 매체를 포함하는 각각의 요리에 coverslips을 전송하기만하면됩니다. 인큐베이터에 다시 이런 요리를 놓습니다. </li><li> 일주일에 한 번, 필요한만큼, 신선한 Neurobasal 매체와 실험 치료와 매체의 3 분의 1을 교체하십시오. 이제 문화에서 이러한 세포의 기능 특성 연구로 준비가되어 있습니다. </li></ol> Hippocampal 뉴런의 Fura - 2 칼슘 이미징 <ol><li> 교양 hippocampal 뉴런 48 시간 (하지만 이전)에 대한 체외에 있었 후, 칼슘 이미징 실험을 시작할 수 있습니다. 이 시점에서,이 뉴런들은 프로세스를 확장하기 시작한다. 칼슘 이미징을위한 최적의 연령 범위는 체외 3 일에서 7입니다. </li><li> Fura - 2와 부하 문화 37 30 분 AM ° 그들이 어떤 샘플 매 150ms를 칼슘 표시기 염료 및 512 비트 CCD 카메라를 자극하는 제논 아크 램프를 사용하여 20X 목적에 따라 몇 군데 수있는 후에 C. </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>

이 프로토콜은 게리 뱅커와 김 고슬린 1의 혁신적인 작품의 수정으로 개발되었습니다. 이 책은 culturing 세포에 관심이 사람을위한 필수적인 리소스입니다 -뿐만 아니라 신경 세포 - 풍부한 문화는 현재 프로토콜 설명했다. 세포 culturing에있는 세 개의 가장 중요한 요소는 다음과 같습니다 불임, 속도, 사용되는 매체의 선택이다. 불임 - 층류 / 살균 후드, 무균 조건 및 무균 인큐베이...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<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>
		</div>

primary culture of hippocampal neurons from p0 newborn rats

hippocampal 신경의 생리적 특성은 일반적으로 특히 때문에 학습 및 메모리에 해마의 참여의 조사입니다. 기본 hippocampal 세포 culturing은 neuroscientists는 개별 세포와 단일 버렸네 수준에서 뉴런의 활동과 속성을 확인하실 수 있습니다. 이 비디오에서는, 우리는 신생아 쥐의 기본 hippocampal 세포를 분리하고 성장하는 방법을 보여줍니다. 해마는 2~3분만큼 짧은 각 신생아 동물로부터 격리 수 있으며, 문화 두 주에 유지하실 수 있습니다. 우리는 또한 간략하게 ratiometric 칼슘 이미징 이러한 hippocampal 뉴런을 사용하는 방법을 보여줍니다 것입니다. 이 프로토콜은 어떠한 수정을 거의, 해마에 대한 과정을 설명하는 동안, 그것은 두뇌의 다른 지역에 적용할 수 있습니다.

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

해부 및 개별 뇌 영역에서 세포의 성장은 세포와 생리적 매개 변수의 조사를 용이하게합니다. 우리는 혈청이없는 환경에서 신경 세포 - 풍부한 문화를 생산하는 주요 세포 culturing에 대한 방법을 설명합니다.

P0 신생아 쥐에서 Hippocampal 신경의 주요 문화

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

76.4K 조회수.  Michigan State University (MSU). 해부 및 개별 뇌 영역에서 세포의 성장은 세포와 생리적 매개 변수의 조사를 용이하게합니다. 우리는 혈청이없는 환경에서 신경 세포 - 풍부한 문화를 생산하는 주요 세포 culturing에 대한 방법을 설명합니다.

비디오: Primary Culture of Hippocampal Neurons from P0 Newborn Rats

Gene-gun Transfection of Hippocampal Neurons

Hippocampal 뉴런의 유전자 총을 Transfection

연구

생물학

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.

기본 교양 Hippocampal 신경에 덴스 코어 Vesicles의 라이브 영상

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.

성인 쥐 심장 Myocytes의 차 문화

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.

성인 마우스 Hippocampal 이가있는 이랑의 해부

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%.

쥐 간장 세포의 분리 및 차 문화

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.

분리 및 마우스 대동맥 내피 세포의 차 문화

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.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

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.

소형 돼지에서 1 차 대동맥 내피 세포의 격리 및 배양

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.

신생아 마우스에서 기본 백색및 갈색 프레드포스세포의 격리 및 분화

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.

성인 마우스 입천장에서 1 차 구강 각막 세포의 격리 및 문화

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