بدعم من : USPHS منح NS38500 ، المعهد الوطني للاضطرابات العصبية والسكتة الدماغية ، المعاهد القومية للصحة بالولايات المتحدة الأمريكية ، التكنولوجيا الطبية الحيوية التحالف جنوب شرق ويسكونسن ، Milwauke ، ويسكونسن ، الولايات المتحدة الأمريكية ؛ بريون Riesch مؤسسة الشلل ، وميلووكي ، ويسكونسن ، الولايات المتحدة الأمريكية ؛ ترتيب الشقيق النسور ، والغرب الأوسط الفصل ، الولايات المتحدة الأمريكية ؛ الوطنية لاصابة النخاع الشوكي الرابطة ، الفصل ميلووكي ، الولايات المتحدة الأمريكية ، شمال شرق انكلترا معهد الجذعية الخليوي ، جامعة نيوكاسل في المملكة المتحدة.

تشريح من الانتفاخ من الكبار المسام الشارب ماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نحن نستخدم 10 أسابيع إلى 6 أشهر من العمر الفئران. الفئران الأصغر الغلة غالبا ما تكون أكثر الخلايا. الموت ببطء والفأر حيوان تغرق بالكامل في خليط من betadine 01:01 (محلول اليود) وبيروكسيد الهيدروجين (المتوفر من الصيدلية) لحوالي 3 دقائق. </li><li style=";text-align:right;direction:rtl"> بخ كامل الماوس ، لا سيما في منطقة الوجه ، مع الايثانول 75 ٪ ، وحمل الماوس لمجهر تشريح هود في التدفق الصفحي. </li><li style=";text-align:right;direction:rtl"> تشريح منصات الطولي ، وتجنب القطع بعناية في بصيلات الشعر ، وحمام سباحة في HBSS. </li><li style=";text-align:right;direction:rtl"> تشريح بصيلات الطولي وحمام سباحة في HBSS في درجة حرارة الغرفة. القيام بذلك عن طريق عقد الجلد بجانب بصيلات الشعر مع ملقط ، ومن ثم قطع جميع أنحاء جراب مع المقص مباشرة. قطع عميق لتجنب اصابة بصيلات الشعر. </li><li style=";text-align:right;direction:rtl"> رفع جريب شعرة شعرة من لوحة ووضعها في لوحة جديدة مع HBSS الطازجة. </li><li style=";text-align:right;direction:rtl"> تدفق adipous فضفاضة والنسيج الجلدي مع النافورات من العازلة ، وحتى المسام نظيفة ضئيلة للغاية. إذا لزم الأمر ، وقطع العصب للجريب شعرة وإزالة الأنسجة الملتصقة عن طريق كشط والهبات المتكررة HBSS. </li><li style=";text-align:right;direction:rtl"> دبوس شعر على تنظيف follilce Sylgard المغلفة الزجاج طبق بتري باستخدام الإبر التنغستن شحذ ، وذلك باستخدام microforceps عن التمسك الجزء الجلد المجاور للجريب. </li><li style=";text-align:right;direction:rtl"> قطع شعرة جراب longitudically مع microblade. تجنب قطع عميق جدا ، حيث سيؤدي ذلك إلى إصابة انتفاخ. إزالة الدم مع النافورات المتكررة HBSS حتى ذهب. ظهور الدم يعتبر مؤشرا جيدا على أن خفض كانت عميقة بما فيه الكفاية. إذا لم يتم خفض longitudial طويلة بما فيه الكفاية ، يجوز إطالتها مع microscissors عازمة. </li><li style=";text-align:right;direction:rtl"> اجراء خفض عرضية فوق مستوى الجيوب الكهفية ، وبعد ذلك بخفض ثان على مستوى عرضية داخل تجويف حلقة ، على مقربة من الجلد. </li><li style=";text-align:right;direction:rtl"> سترى الآن انتفاخ داخل الكبسولة. </li><li style=";text-align:right;direction:rtl"> الاستيلاء على وضع حد لكبسولة من الكولاجين مع ملقط وطرح انتفاخ بإبرة التنغستن عازمة. سترى الآن الكبسولة الفارغة وانتفاخ معزولة. </li><li style=";text-align:right;direction:rtl"> عزل تجمع الانتفاخات في صفيحة ثقافة منفصلة في HBSS في درجة حرارة الغرفة. تأكد من عدم الأنسجة الأخرى هي تلوث الانتفاخات المجمعة. </li></ol> ثقافة Explants انتفاخ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> معطف 35 ملم مع لوحات ثقافة الكولاجين الكولاجين من خلال وضع 50 و 10 ميكرولتر ميكرولتر العقيمة كلوريد الصوديوم 6 ٪ بجانب بعضها البعض. تخلط جيدا مع الماصة Pasteyr المزدوج عازمة والدفع باتجاه حافة صفيحة الثقافة. يحضن بين عشية وضحاها في dessicator نظيفة ، ولكن لا تدع الجافة. قبل الاستخدام ، لوحات شطف مع المياه المالحة. </li><li style=";text-align:right;direction:rtl"> قبل احتضان الثقافة لوحات الكولاجين المغلفة وتشطف لحوالي 3 دقائق مع متوسط ​​الثقافة. مستنبت يتكون من 85 ٪ الفا تعديل MEM ، و 10 ٪ مصل بقري جنيني و 5 ٪ اليوم استخراج الجنين الفرخ 11. </li><li style=";text-align:right;direction:rtl"> بعد إعادة حضانة ، وإزالة لوحات مستنبت من الانتفاخات وإضافة عدة مع متوسط ​​أقل قدر ممكن. إزالة الزائدة متوسطة الثقافة. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 1 ساعة ، ولكن لم يعد ، في الخلية الحاضنة في جو مرطب مع الأكسجين 10 ٪ و 5 ٪ CO 2. </li><li style=";text-align:right;direction:rtl"> بعد 1 ساعة ، إضافة بلطف 1.5 مل من المتوسط ​​الثقافة. ينبغي explants انتفاخ التمسك التحتية الكولاجين. استبدال 50 ٪ من متوسط ​​الثقافة اليومية. </li><li style=";text-align:right;direction:rtl"> في غضون 3 -- 4 أيام ، وسوف الخلايا الكثيرة الارتحال الهجرة من explants انتفاخ. علما التشكل على نجمي ، حركية ، وعدم وجود الغالبة من خلية خلية الاتصالات. خلال الأيام القليلة المقبلة ، سيكون أكثر الخلايا الهجرة ، وهاجر وسوف تتكاثر الخلايا بسرعة. </li><li style=";text-align:right;direction:rtl"> إزالة انتفاخ 2 -- 3 أيام بعد ظهور EPI - NCSC الهجرة بإبرة التنغستن شحذ. إذا ترك الانتفاخات أطول ، فإنها تميل إلى تسطيح ويجعل من المستحيل الحصول على برنامج التحصين الموسع ، نقية NCSC الثقافات. </li><li style=";text-align:right;direction:rtl"> المهم : خلايا نادرة مع التشكل بالارض ، والتي أصبحت واضحة في بعض الأحيان عدة أيام في وقت لاحق من البرنامج الموسع للتمنيع ، NCSC ، والتي هي أقل متحركة وغير EPI - NCSC ، ولكن المفترض الخلايا الجذعية البشرة / الأسلاف. الثقافات التي تتكون من هذه الخلايا ، أو الثقافات المختلطة التي تحتوي على برنامج التحصين الموسع ، والمفترض NCSC الخلايا الجذعية البشرة / الأسلاف يجب أن يتم التخلص منها. </li><li style=";text-align:right;direction:rtl"> تلميح : لا تبقي الخلايا لفترة طويلة جدا في المتوسط ​​يزدرع الابتدائية ، كما أنها تميل إلى التفريق في كثافة الخلايا بسرعة عالية ، وبسبب مزعومة autocrine / نظير الصماوي النمو يشير عامل. </li></ol>

<ol>
	<li>Sieber-Blum, M., Grim, M., YF, H. u., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Sieber-Blum, M., Grim, M., Tuan, R., Grim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+hair+follicle+-+cradle+for+pluripotent+neural+crest+stem+cells.">The adult hair follicle - cradle for pluripotent neural crest stem cells.</a> Birth Defects Research (Part C). 72, 162-172 (2004).</li><li>Sieber-Blum, M., Schnell, L., Grim, M., Schneider, R., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+epidermal+neural+crest+stem+cell+(EPI-NCSC)+grafts+in+the+lesioned+spinal+cord.">Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.</a> Pub. 32, 67-81 (2006).</li><li>Hu, Y. F., Zhang, Z. -. J., Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epidermal+neural+crest+stem+cell+(EPI-NCSC)+molecular+signature.">An epidermal neural crest stem cell (EPI-NCSC) molecular signature.</a> Stem Cells. 24, 2692-2702 (2006).</li></ol>

بحكم قدرتها على الهجرة ، ويمكن أن تكون معزولة EPI - NCSC كما يبلغ عدد سكانها نقية للغاية من الخلايا الجذعية ، التي يمكن توسيعها في المختبر. كبقايا الجنينية في موقع الكبار ، EPI - NCSC المرشحين يحتمل أن تكون جذابة بالنسبة لمستقبل العلاجات البديلة للخلية ، الهندسة الطبية و / أو الطب التجدي?...

mouse epidermal neural crest stem cell (epi-ncsc) cultures

EPI - NCSC هي من مخلفات العصبية الجنينية للقمة في مكان غير أخلاقي ، وانتفاخ في بصيلات الشعر. هم الخلايا الجذعية multipotent التي لديها خاصية فسيولوجية لتوليد مجموعة واسعة من أنواع الخلايا المختلفة ، بما في ذلك الخلايا العصبية ، الخلايا العصبية الداعمة ، وخلايا العضلات الملساء والعظام / خلايا الغضاريف والخلايا الصباغية. EPI - NCSC يمكن الحصول عليها بسهولة في الجلد وشعر كما يمكن عزل السكان نقية للغاية من الخلايا الجذعية. هذا الفيديو يوفر بروتوكول مفصلة لإعداد الماوس EPI - NCSC الثقافات من بصيلات ضئيلة للغاية. إزالة لوحة من الخط الطولي ماوس الكبار ، وتشريح بصيلات الطولي. المسام تقطع طوليا ثم في وقت لاحق وبشكل مستعرض فوق وتحت منطقة انتفاخ. إزالة انتفاخ من كبسولة الكولاجين وضعت في لوحة الثقافة. EPI - NCSC بدء الهجرة من انتفاخ explants 3 إلى 4 أيام في وقت لاحق.

Watch this Scientific Journal Video about Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures at JoVE.com

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

نعرض هنا لدينا وسيلة لعزل الماوس البشرة الجذعية العصبية قمة الخلايا (EPI - NCSC). الأسلوب الذي ينطوي على تشريح المسام الدقيقة ضئيلة للغاية ، وعزل انتفاخ placeing عليه في زراعة الأنسجة. EPI - NCSC بدء الهجرة من explants انتفاخ على الطبقة التحتية في غضون 3 -- 4 أيام.

الماوس البشرة الجذعية العصبية خلية كريست (EPI - NCSC) الثقافات

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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Biology

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

11.5K Views.  Newcastle University. نعرض هنا لدينا وسيلة لعزل الماوس البشرة الجذعية العصبية قمة الخلايا (EPI - NCSC). الأسلوب الذي ينطوي على تشريح المسام الدقيقة ضئيلة للغاية ، وعزل انتفاخ placeing عليه في زراعة الأنسجة. EPI - NCSC بدء الهجرة من explants انتفاخ على الطبقة التحتية في غضون 3 -- 4 أيام.

Video: Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

ثقافة العصبية الماوس السلائف الخلايا الجذعية

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

تشريح 6.5 أجنة الفئران DPC

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

عد الخلايا الجذعية العصبية البشرية

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

الركض الخلايا الجذعية العصبية البشرية

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

كيمياء سيتولوجية مناعية : الخلايا الجذعية العصبية البشرية

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

تقييم العصبية على الحركة باستخدام الخلايا الجذعية جل المستندة Agarose الأجهزة ميكروفلويديك

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

جيل وحيدة الخلية معلقات من الأنسجة العصبية ماوس

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

إعداد Lysate مجمع صفائح الدم البشرية (pHPL) باعتبارها الملحق لمصل فعال خالية من الإنسان الحيوان الثقافات الخلايا الجذعية

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

يعيش التصوير في القدرة على الحركة والهيكل الخلوي أكتين خلية من الخلايا العصبية الفردية والخلايا العصبية في الأجنة كريست اسماك الزرد

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

إزالة الخلايا الليفية السريع من الكثافة البشرية العالية الجنينية الخلايا الجذعية الثقافات

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...