وأيد JLN بواسطة MH 68347.

قبل قرن آمون إلى العزلة قبل البدء في عزلة الحصين ، تأكد من أن جميع الأدوات معقمة. رذاذ أسفل غطاء الثقافة مع الايثانول 70 ٪ ، ووضع الأدوات داخل غطاء محرك السيارة. ستحتاج 6 -- و 10 سم ، أطباق بتري ، معقمة بولي - L coverslips الزجاج المطلي ، وpipettors نصائح ، ماصات القابل للتصرف ، وpipettor الكهربائية. من هذه النقطة ، تذكر أن استخدام تقنية معقمة الصحيح. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بدوره على حمام مائي وتأكد من أن يتم تسخينها إلى 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> الحلول التالية التي يتم تخزينها في 4 درجات مئوية وستكون هناك حاجة : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعديل النسر متوسطة (MEM) </li><li style=";text-align:right;direction:rtl"> Neurobasal </li><li style=";text-align:right;direction:rtl"> هانك محلول ملحي مخزنة (HBSS) </li><li style=";text-align:right;direction:rtl"> بورات العازلة الحل </li><li style=";text-align:right;direction:rtl"> الصوديوم البيروفات الحل </li><li style=";text-align:right;direction:rtl"> العقيمة ، التي تمت تصفيتها حل الجلوكوز 20 ٪ في ماء مقطر </li><li style=";text-align:right;direction:rtl"> تأكد من رش جميع زجاجات أسفل مع الايثانول 70 ٪ قبل وضعها في غطاء محرك السيارة. </li></ul></li><li style=";text-align:right;direction:rtl"> تأخذ هذه الحلول المجمدة من الفريزر ووضعها في حوض الماء الساخن : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> A قسامة 5 مل من مصل الحصان </li><li style=";text-align:right;direction:rtl"> A قسامة 1 مل من B - 27 تكملة </li><li style=";text-align:right;direction:rtl"> A قسامة 1 مل من المضادات الحيوية 100X (البنسلين زائد الستربتومايسين) </li><li style=";text-align:right;direction:rtl"> وقسامة 0.5 مل من الجلوتامين - L </li></ul></li><li style=";text-align:right;direction:rtl"> بعد أن تحسنت الحلول ، وتأخذهم بعيدا عن حمام ماء ، رش عليهم مع الايثانول 70 ٪ ، ووضعها في غطاء محرك السيارة. ويمكن الآن أن أعد والمتوسطة Neurobasal الطلاء. </li><li style=";text-align:right;direction:rtl"> بعد مستعدون للحلول ، وكأب الحاويات بإحكام ووضعها في الحمام 37 درجة مئوية في عملية الاحماء أثناء تنفيذ العزلة الحصين. </li><li style=";text-align:right;direction:rtl"> أيضا ، يأخذ قسامة من التربسين البروتيني (2.5 ٪) من الثلاجة وضعها في حمام ماء. سوف تربسين هضم تشريح الحصين ، والتي ستكون معزولة في الخطوة التالية. </li><li style=";text-align:right;direction:rtl"> اخذت انبوب 15ml المخروطية وملئه مع HBSS ، وتسميته مع مجموعة العلاج. ومن هنا أنه سيتم جمعها معزولة الحصين في الخطوة التالية. </li></ol> الحصين العزل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> للبدء في عزلة الحصين ، تأكد من الجراء الوليد نظيفة وكان الفرق حليبها ، مشيمة ، وإزالة الحبل السري من قبل الأم. </li><li style=";text-align:right;direction:rtl"> وضع الجراء في طبق بتري ، مع رش الإيثانول 70 ٪ لتنظيف ، ووضعها في غطاء محرك السيارة. يصرح باستخدامها مباشرة قبل استزراع الحيوانات. </li><li style=";text-align:right;direction:rtl"> لمزيد من التعقيم تأخذ واحدة من الطبق الجرو ، وتراجع في الجرو إلى إيثانول 70 ٪ ، ومن ثم الى اثنين من يغسل HBSS العقيمة. </li><li style=";text-align:right;direction:rtl"> إزالة الرأس من الجسم مع المقص. باستخدام مقص نفسه ، خفضت من خلال الجلد والجمجمة. </li><li style=";text-align:right;direction:rtl"> باستخدام زوج من ملاقط غرامة ، وقشر الجمجمة بعيدا عن الدماغ والمخ في مكان طبق بتري الصغيرة التي تحتوي على كمية صغيرة من HBSS العقيمة. </li><li style=";text-align:right;direction:rtl"> قشر العودة نصفي الكرة المخية. قرن آمون هو صغير ، وفرس البحر على شكل هيكل في الفص الصدغي الإنسي. </li><li style=";text-align:right;direction:rtl"> إزالة الحصين والمكان الى 3 مل من HBSS في أنبوب 15 مل. كرر هذه الخطوات مع كل الجرو ، ومكان كل الحصين معزولة في أنبوب 15 مل. الآن ، حان الوقت لفصل الأنسجة إلى خلايا واحد. </li></ol> الحصين الخلية تفارق <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد أن تم عزل جميع الحصين ، وملء الأنبوب مل 15-4،5 مل مع HBSS. </li><li style=";text-align:right;direction:rtl"> إزالة التربسين من الحمام المائي ، مع رش الإيثانول ، ومكانته في غطاء محرك السيارة. إضافة 0.5 مل من التربسين إلى أنبوب ، واحتضان لمدة 15 دقيقة عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> في غطاء محرك السيارة ، إزالة حل HBSS / التربسين من أنبوب مع ماصة معقمة ، والحرص على عدم تعكير صفو الحصين أن استقروا في الجزء السفلي من الأنبوب. إضافة 5 مل من HBSS إلى الأنابيب ودوامة بلطف. احتضان عند 37 درجة مئوية لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> كرر هذه الخطوة مرتين ، وإزالة HBSS القديمة واستبدالها مع الحل الطازجة. </li><li style=";text-align:right;direction:rtl"> يأخذ قسامة من الدناز خرجت من الثلاجة. إضافة 0.5 مل من الدناز إلى الحصين في HBSS مل 4.5. إضافة إلى تشجيع إهماد الدناز الانزيم. </li><li style=";text-align:right;direction:rtl"> متمزج الحل (أو ماصة صعودا وهبوطا) حتى يصبح متجانس. يجب الحرص على عدم إدخال فقاعات في جناسة. </li><li style=";text-align:right;direction:rtl"> تحديد قابلية الخلية مع استبعاد أسلوب التريبان الأزرق. </li><li style=";text-align:right;direction:rtl"> تأخذ طبق بيتري 10 سم تحتوي على 6-8 coverslips ، وإضافة 10 مل من المتوسط ​​الطلاء. ماصة العدد المطلوب من الخلايا التي تحتوي على الطبق coverslips. دوامة برفق لتفريق الخلايا ، وتأكد من عدم تداخل coverslips. </li><li style=";text-align:right;direction:rtl"> السماح للخلايا تعلق لمدة 2-4 ساعات في ترطيب حاضنة 37 درجة مئوية مع نسبة 5 ٪ CO 2. بعد التأكد من أن الخلايا هي قابلة للتطبيق ويكون المرفقة ، نقل coverslips إلى الأطباق التي تحتوي على الفرد المتوسط ​​Neurobasal. مكان هذه الأطباق العودة إلى الحاضنة. </li><li style=";text-align:right;direction:rtl"> مرة واحدة في الأسبوع ، واستبدال ثلث المتوسط ​​مع المتوسطة Neurobasal الطازجة وعلاجات تجريبية ، حسب الحاجة. الآن ، والخصائص الفنية لهذه الخلايا في الثقافة على استعداد لدراستها. </li></ol> Fura - 2 التصوير الكالسيوم من الخلايا العصبية قرن آمون <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد قرن آمون العصبية مثقف تم في المختبر لمدة 48 ساعة (ولكن ليس في وقت سابق) ، يمكن أن تبدأ تجارب التصوير الكالسيوم. عند هذه النقطة ، يجب أن تبدأ هذه الخلايا العصبية لتوسيع العمليات. الفئة العمرية الأمثل للتصوير من الكالسيوم يوميا في المختبر 3-7. </li><li style=";text-align:right;direction:rtl"> تحميل مع الثقافات Fura - 02:00 لمدة 30 دقيقة عند 37 درجة مئوية ، وبعد ذلك يمكن تصوير ما تحت الهدف 20X باستخدام مصباح قوس زينون لإثارة صبغ المؤشر الكالسيوم وكاميرا CCD 512 بت ، والتي العينات كل 150ms. </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>

وقد تم تطوير هذا البروتوكول بمثابة تعديل للعمل منوية من بانكر غاري وكيم Goslin 1. هذا الكتاب هو موردا أساسيا للراغبين في زراعة الخلايا -- وليس فقط الثقافات عصبون التخصيب هو موضح في البروتوكول الحالي. العوامل الثلاثة الأكثر أهمية في استنبات الخلايا هي...

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<td>Antibiotic antimitotic</td>
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<td>15240062</td>
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<td>B-27 Supplement</td>
<td>&nbsp;</td>
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<td>17504044</td>
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<td>Boric Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B6768</td>
<td>&nbsp;</td>
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<td>Dnase I</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>DN25</td>
<td>&nbsp;</td>
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<td>F1221</td>
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<td>Poly-L-Lysine Hydrobromide</td>
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<td>P6282</td>
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<td>Sigma</td>
<td>P2256</td>
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<td>P2256</td>
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<td>Invitrogen</td>
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primary culture of hippocampal neurons from p0 newborn rats

عادة يتم التحقيق الخصائص الفيزيولوجية للعصبونات الحصين ، وخصوصا بسبب تورط حصين في التعلم والذاكرة. زراعة الخلايا الأولية الحصين يسمح علماء الأعصاب على دراسة النشاط وخصائص الخلايا العصبية في زنزانة فردية واحدة على مستوى المشبك. في هذا الفيديو ، سوف نقوم شرح كيفية عزل وزراعة الخلايا الأولية الحصين من الفئران حديثي الولادة. قد تكون معزولة الحصين من كل حيوان الأطفال حديثي الولادة في قصيرة بقدر 2 إلى 3 دقائق ، ويمكن المحافظة على الثقافات لمدة تصل الى أسبوعين. ونحن أيضا لفترة وجيزة تبين كيفية استخدام هذه الخلايا العصبية الحصين للتصوير ratiometric الكالسيوم. في حين أن هذا البروتوكول يصف عملية للقرن آمون ، مع قليل من دون تعديل ، ويمكن تطبيقه في مناطق أخرى من الدماغ.

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

تشريح ونمو الخلايا من منطقة في الدماغ الفردية يسهل التحقيق في المعلمات الخلوية والفسيولوجية. نحن تصف طريقة لزرع الخلايا الأولية التي تنتج الخلايا العصبية التخصيب الثقافات في بيئة خالية من المصل.

الثقافة الأولية من الخلايا العصبية قرن آمون من الفئران حديثي الولادة P0

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 Views.  Michigan State University (MSU). تشريح ونمو الخلايا من منطقة في الدماغ الفردية يسهل التحقيق في المعلمات الخلوية والفسيولوجية. نحن تصف طريقة لزرع الخلايا الأولية التي تنتج الخلايا العصبية التخصيب الثقافات في بيئة خالية من المصل.

Video: Primary Culture of Hippocampal Neurons from P0 Newborn Rats

Gene-gun Transfection of Hippocampal Neurons

مدفع الجينات ترنسفكأيشن من الخلايا العصبية قرن آمون

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.

يعيش تصوير الكثيفة الأساسية الحويصلات في الخلايا العصبية الأولية الحصين مثقف

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.

الابتدائية ثقافة الكبار Myocytes قلب فأر

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.

تشريح التلفيف المسنن من الحصين ماوس الكبار

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%.

العزلة والثقافة الأساسي في الخلايا الكبدية الجرذ

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.

العزلة والثقافة الابتدائية من خلايا الفأر الأورطي غشائي

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.

عزل وثقافة الكرياتينية الماوس الرئيسية من حديثي الولادة والفأر الكبار الجلد

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.

عزل وثقافة الخلايا البطانية الأبهرية الأولية من الخنازير المصغرة

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.

عزل وتمايز الخلايا التمهيدية الأولية البيضاء والبنية من الفئران حديثي الولادة

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.

عزل وثقافة الكريات الكيراتينية الفموية الأولية من الحنك الفأري البالغ

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