صنع رصاصات بندقية الجينات (GOLD) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل 100X PVP (0.5mg/ml) في EtOH 100 ٪. </li><li style=";text-align:right;direction:rtl"> تزن خارج 30mg من 1.6 ميكرون microcarrier BioRad الذهب ووضعه في أنبوب 1.5 مل إيبندورف. </li><li style=";text-align:right;direction:rtl"> حساب كمية البلازميد اللازمة لإعطاء تركيز الذهب البلازميد 0،5-3 ميكروغرام / ملغ ، ووضع البلازميد في أنبوب مع microcarriers الذهب. الجمع بين عدة البلازميدات إذا لزم الأمر. </li><li style=";text-align:right;direction:rtl"> جلب حجم البلازميد الى 100 ميكرولتر مع الماء المقطر. </li><li style=";text-align:right;direction:rtl"> إضافة 100 ميكرولتر من سبيرمدين 0.05M في الأنبوب. دوامة أنبوب 30 ثانية. </li><li style=";text-align:right;direction:rtl"> دوامة 1 دقيقة بسرعة كاملة. يصوتن لمدة 3-5 دقائق.. </li><li style=";text-align:right;direction:rtl"> جافة طول المناسب للأنابيب Tefzel في جهاز PDS-1000/He Biorad لمدة 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> بعد صوتنة ، لمدة 1-2 دقائق دوامة بأقصى سرعة. خفض السرعة ببطء للسماح بفتح أنبوب بينما vortexing. </li><li style=";text-align:right;direction:rtl"> إضافة 100 ميكرولتر من CaCl M 1 2 ببطء إلى الانخفاض في حين vortexing أنبوب الحكيمة. </li><li style=";text-align:right;direction:rtl"> ترك الأنبوب في درجة حرارة الغرفة لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> جعل 5 ميكرون تصفية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يستغرق عامين المحاقن 5ml دون الإبر. </li><li style=";text-align:right;direction:rtl"> إزالة المكبس ، وفصل القبعات السوداء. أخذ مقص صغير ، وقطع جزء من الغطاء الأسود لإعطاء الحلبة. كرر على المكبس أخرى لالحلقتين الصافية. </li><li style=";text-align:right;direction:rtl"> شطيرة مرشح 5 ميكرون (أجزاء صغيرة ، وشركة النايلون التصفية ، ونصف قطرها بوصة) بين الحلقتين ، ودفع شطيرة في رأس المحقنة. </li><li style=";text-align:right;direction:rtl"> حقنة تتدلى على قارورة 50ml المخروطية. </li></ol></li><li style=";text-align:right;direction:rtl"> بعد 10 دقيقة ، ودوامة أنبوب لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> أنبوب الطرد المركزي لمدة 30 ثانية ، وحفظ بيليه. </li><li style=";text-align:right;direction:rtl"> إضافة 1ml من EtOH 100 ٪ لبيليه. استخدام غيض ماصة لتفريق بيليه ، والدوامة 30 ثانية. تدور 30 ​​ثانية ، وإزالة EtOH. 2 كرر أكثر من مرة. </li><li style=";text-align:right;direction:rtl"> إضافة 1ml من EtOH 100 ٪ لبيليه (فاصل مع الطرف). هذا المكان في تصفية 5 ميكرون والسماح لها من خلال تدفق عن طريق الجاذبية في أنبوب 50ml المخروطية. EtOH استخدام أكثر حسب الحاجة لغسل خارج الأنبوب أو من خلال المساعدة في التدفق. </li><li style=";text-align:right;direction:rtl"> تدور في أنبوب 50ml 2000rpm في أجهزة الطرد المركزي لمدة 5-10 دقائق. إزالة طاف ، حفظ بيليه. </li><li style=";text-align:right;direction:rtl"> اعتمادا على الكثافة المطلوبة من microcarriers الحاجة ، إضافة 800μl - 1.5ml EtOH 100 ٪ و 8 - 15 ميكرولتر المقابلة حل PVP ميكرولتر. </li><li style=";text-align:right;direction:rtl"> دوامة وانتقل على الفور الى جهاز PDS-1000/He Biorad. </li><li style=";text-align:right;direction:rtl"> Biorad جهاز PDS-1000/He <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع الانبوب الذي يربط خزان الغاز في أنبوب N2 التجفيف </li><li style=";text-align:right;direction:rtl"> إخراج التجفيف أنابيب Tefzel من الجهاز </li><li style=";text-align:right;direction:rtl"> إدراج أحد طرفيه في أنبوب 50ml المخروطية ، وإرفاق حقنة إلى الطرف الآخر من الأنبوب. تمتص الطين البني في وسط الأنبوب. </li><li style=";text-align:right;direction:rtl"> العودة الى ادخال أنابيب آلة. </li><li style=";text-align:right;direction:rtl"> الانتظار (بدون التناوب) لمدة 4 دقائق </li><li style=";text-align:right;direction:rtl"> بعد 4 دقائق ، تمتص قبالة EtOH ببطء وبعناية ، والتأكد من عدم الإخلال microcarriers. </li><li style=";text-align:right;direction:rtl"> تدوير أنابيب لمدة 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> بعد 1 دقيقة ، وتوصيل أنبوب يربط خزان غاز N 2 إلى أنبوب التجفيف والسماح أنابيب جافة لمدة 15 دقيقة. </li></ol></li><li style=";text-align:right;direction:rtl"> بعد 15 دقيقة ، وقطع الأنابيب لحجم رصاصة لاستخدامها مباشرة أو تخزينها في 4 درجات مئوية في exsiccator. ويمكن تخزين الرصاص تصل إلى 3 أشهر. </li></ol> إعداد مدفع الجينات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الرصاص في مكان خرطوشة. ويمكن اطلاق النار كل قطعة من شبكية العين مع الرصاص 1-3. </li><li style=";text-align:right;direction:rtl"> إدراج خرطوشة بندقية في الجينات ونعلق البندقية إلى مصدر الهليوم. تعيين الضغط الى 110 رطل. </li></ol> إعداد وسائل الاعلام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تشريح المتوسطة <table style="width: 229px; height: 157px;;text-align:right;direction:rtl" border="0"><tbody><tr><td> 1 زجاجة أميس الحل (سيغما ، 8.8g) </td></tr><tr><td> بيكربونات الصوديوم 1.9 غم </td></tr><tr><td> 10 مل القلم 1 ٪ / بكتيريا / L - الجلوتامين (1:100) (Gibco / Invitrogen) </td></tr><tr><td> 990 مل ماء مقطر ل1L </td></tr><tr><td> ------------------------------- </td></tr><tr><td> 1 ليتر </td></tr></tbody></table> </li><li style=";text-align:right;direction:rtl"> تصفية المتوسطة تشريح باستخدام تصفية 0.22 ميكرون. </li><li style=";text-align:right;direction:rtl"> متوسطة الحضانة (500ml) <table style="width: 283px; height: 157px;;text-align:right;direction:rtl" border="0"><tbody><tr><td> 485 مل من وسائل الاعلام التشريح التي تمت تصفيتها </td></tr><tr><td> 5 مل من تكملة N2 1 ٪ (Gibco / Invitrogen) </td></tr><tr><td> 1 ٪ 10ML مصل الحصان (سيجما) </td></tr><tr><td> 500 ميكرولتر من أحمر الفينول </td></tr><tr><td> -------------------------------------------------- -- </td></tr><tr><td> 0.5 لتر </td></tr></tbody></table> </li><li style=";text-align:right;direction:rtl"> تصفية المتوسطة حضانة باستخدام تصفية 0.22 ميكرون. </li><li style=";text-align:right;direction:rtl"> فقاعة O 2 في تشريح المتوسطة لمدة 15 دقيقة قبل بدء الحصاد بروتوكول الشبكية. </li></ol> إعداد غرف الحضانة لشبكية العين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحت غطاء محرك السيارة ، وملء 60 ملم عدة أطباق عميقة (غرف الحضانة ، نونك) مع 25 مل من المتوسط ​​الحضانة. عدد من الأطباق يعتمد على عدد من عطنإيناس اللازمة. </li><li style=";text-align:right;direction:rtl"> مكان موقف مرشح في كل صحن عميق. وينبغي أن لا يقتصر على نصائح جدا من موقف أن تغمرها المياه. </li><li style=";text-align:right;direction:rtl"> وضع جميع الأطباق في عمق الحاضنة حتى شبكية العين جاهزة. </li><li style=";text-align:right;direction:rtl"> ملء two 100 ملم أطباق بتري وتقديمهم إلى مقاعد البدلاء تشريح. </li></ol> التحضير للشبكية الأرنب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التضحية أرنب وفقا لبروتوكولات المعمول بها. التالية لا يجب أن يتم تحت غطاء محرك السيارة. </li><li style=";text-align:right;direction:rtl"> مع مقص الزاوية ، أدخله وراء العين في تجويف العين من الأرانب ، وقطع الأغشية والأعصاب البصرية التي تعلق على العين. </li><li style=";text-align:right;direction:rtl"> رفع بلطف خارج العين وغسله مع وسائل الاعلام تشريح الاوكسيجين. </li><li style=";text-align:right;direction:rtl"> مكان العين (القرنية مواجهة) في البئر لتقطيع العين وإغلاق الغطاء. </li><li style=";text-align:right;direction:rtl"> ضع شفرة الحق أفقيا تحت الغطاء. في اقتراح واحد على نحو سلس ، واستخلاص عبر النصل العين لقطع القرنية ، مما يترك كوب العين. </li><li style=";text-align:right;direction:rtl"> باستخدام ملقط ، إزالة شبكية العين من تقطيع اللحم جيدا ووضعه على ورقة التصفية. </li><li style=";text-align:right;direction:rtl"> إزالة العدسة والجسم الزجاجي التي لقط منطقة قريبة من العصب البصري وقطعت وسحب إلى الوراء على ورق الترشيح. كرر عدة مرات حتى يتم إزالة ما يقرب من جميع الزجاجي ، ويتطلع العين بددت الكأس. </li><li style=";text-align:right;direction:rtl"> مكان كوب العين في طبق بتري مع وسائل الإعلام لغسل تشريح. </li><li style=";text-align:right;direction:rtl"> قطع كوب العين في العدد المرغوب فيه من قطعة (5 قطع كحد أقصى). </li><li style=";text-align:right;direction:rtl"> أخذ قطعة واحدة من كوب العين ووضعه تحت المجهر. </li><li style=";text-align:right;direction:rtl"> المشبك والصلبة والمشيمية معا برفق على حافة الشبكية باستخدام ملقط. مع من ناحية أخرى ، واستخدام فرشاة لتنظيف غرامة الصحيح بين المشيمية والشبكية. </li><li style=";text-align:right;direction:rtl"> باستخدام حركات قصيرة لطيف ، ندف الشبكية قبالة المشيمية حتى يصبح منفصلة. </li><li style=";text-align:right;direction:rtl"> قطع 1 بوصة قبالة ماصة نقل معقمة. استخدام هذا لتمتص شبكية العين والمكان الى طبق بتري المتوسطة حضانة لغسل. </li><li style=";text-align:right;direction:rtl"> باستخدام آخر ماصة نقل معقمة مع 1 بوصة قطع ، بعناية نقل الشبكية على مرشح ميكرومتر Millicell 0.4 (ميليبور لعدم وجود القطة : PICMORG50). </li><li style=";text-align:right;direction:rtl"> استخدام الفرشاة لشبكية العين وتوجيه العقدة مع الجانب الخلية التي تواجه صعودا. </li><li style=";text-align:right;direction:rtl"> تتسطح خارج شبكية العين بواسطة بالفرشاة برفق على حواف الشبكية. تقليل لمس طبقة الخلايا العقدية مع فرشاة قدر الإمكان. </li><li style=";text-align:right;direction:rtl"> إزالة الزائدة المتوسطة على تصفية مع نقل ماصة. </li><li style=";text-align:right;direction:rtl"> باستخدام الملقط ، ونقل التصفية الصعود إلى suctioner تعديلها. الاعتماد على suctioner 2-3 مرات لإزالة كافة المتوسطة الحضانة من مرشح. </li></ol> العدة الجينات وحضانة للشبكية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحت غطاء محرك السيارة ، ومكان كل واحدة من المرشحات التي تحتوي على شبكية العين ، على المدرجات التصفية في أطباق عميقة 60 ملم (غرف الحضانة). </li><li style=";text-align:right;direction:rtl"> تأكد من أن المتوسط ​​هو على اتصال مع الجانب السفلي من التصفية ، وأنه وسيلة لا تمتد على جانبي فلتر على شبكية العين. ينبغي للمرشح بمثابة واجهة بين شبكية العين والمتوسطة. </li><li style=";text-align:right;direction:rtl"> اطلاق النار على كل قطعة من شبكية العين مع اثنين من الرصاص. مكان مدفع الجينات مباشرة فوق الأنسجة ، وحوالي 3-4 سم بعيدا. </li><li style=";text-align:right;direction:rtl"> إخضاع جميع غرف الحضانة في الصعود إلى شاكر في 35-37 درجة مئوية الحاضنة. </li><li style=";text-align:right;direction:rtl"> استبدال المتوسطة حضانة في كل يوم. يمكن المحتضنة في الشبكية لمدة أقصاها 6 أيام. </li></ol>

<ol>
	<li>Landreth, . B. W., Agranoff, N. R., Smalheiser, S. M., Crain, M. B., Bornstein, R., Adler, P. J., Magistretti, A. G., Hyndman, W. J., Shoemaker, H., Rehm, T., Schafer, H., Betz, S. U., Kim, H., Takahashi, A. R., Caffe, A., Soderpalm, T., van Veen, H. C., Wong, S., Thompson, D. Y., Yu, S. J., Cringle, V. A., Alder, S. J., Taylor, G. M., Seigel, A. R., Caffe, P., Ahuja, B., Holmqvist, S., Azadi, J., Forsell, I., Holmqvist, J., Hatakeyama, R., Kageyama, S. S., Zhang, X. Y., Fu, C. J., Barnstable, S. W., Wang, X., Mu, W. J., Bowers, W. H., Klein, T., Inoue, M., Hojo, Y., Bessho, Y., Tano, J., Lee, R., Kageyama, A., Kretz, S. H., Hermening , S., Isenmann , I., Liljekvist-Larsson, K., Johansson, B., Reidel, W., Orisme, T., Goldmann, W. C., Smith, U., Wolfrum, H., Xin, J. A., Yannazzo, R. S., Duncan, E. V., Gregg, M., Singh, P., Koulen, S. A., Johnston, D. C., Tang, D. C., Lo, A. K., McAllister, K. a. t. z., LC, L. E., Rajagopalan, J. S., Malter, A. K., McAllister, W. -. B., Gan, J., Grutzendler, W., Wong, R., Wong, J., Lichtman, H., Sato, S., Hattori, S., Kawamoto, I., Kudoh, A., Hayashi, I., Yamamoto, G., Zhang, M. E., Selzer, R. L., Sohn, M. T., Murray, K., Schwarz, J., Nyitray, P., Purray, A. P., Franko, J. A., O'Brien, M., Holt, G., Whiteside, S. C., Lummis, M. H., Hastings, L., Klimaschewski, W., Nindl, M., Pimpl, P., Waltinger, K., Pfaller, P., Kettunen, J., Demas, C., Lohmann, N., Kasthuri, Y., Gong, R. O., Wong, N., Gamper, M. S., Shapiro, M. J., Wirth, P., Wahle, J., O'Brien, S. C., Lummis, A. M., Benediktsson, S. J., Schachtele, S. H., Green, M. E., Dailey, L., Chow, E. M., Levine, T. A., Reh, R. L., Rockhill, F. J., Daly, M. A., MacNeil, S. P., Brown, R. H., Masland, W., Sun, N., Li, S., He, W., Sun, N., Li, S., He, R. C., Stacy, R. O., Wong, E., Strettoi, V., Pignatelli, C., Rossi, V., Porciatti, B., Falsini, V., Pignatelli, E., Strettoi, V. P., Connaughton, D., Graham, R., Nelson, P. H., Edqvist, F., Hallbook, T. C., Jakobs, R. T., Libby, Y., Ben, S. W., John, R. H., Masland, Y., Qin, G., Xu, W., Wang, R., Roizenblatt, J. D., Weiland, S., Carcieri, G., Qiu, M., Behrend, M. S., Humayun, A., Koizumi, G., Zeck, Y., Ben, R. H., Masland, T. C., Jakobs, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture+of+adult+goldfish+retina:+a+model+for+the+study+of+CNS+regeneration.">Explant culture of adult goldfish retina: a model for the study of CNS regeneration.</a> Brain Res. 161, 39-55 (1979).</li></ol>

1. ماذا يمكنني أن أفعل مع هذا الأسلوب؟ <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> انظر مورفولوجيا الخلايا بشكل واضح. </li><li style=";text-align:right;direction:rtl"> تسمية الخلايا ليسجل منها. </li><li style=";text-align:right;direction:rtl"> Overexpress البروتينات ، مثل PSD95 ، ولكن أيضا البروتينات الأخرى التي تعدل استجاب...

organotypic culture of adult rabbit retina

نظم الثقافة عضوي النمط من الأنسجة العصبية الوظيفية هي أدوات هامة في مجال البحوث العصبية الحيوية. من الناحية المثالية ، ينبغي أن يكون هذا النظام متوافق مع تقنيات التصوير ، والتلاعب الجيني ، وتسجيل الكهربية. هنا نقدم نسيج الطور البيني نظام بسيط للثقافة الشبكية الأرانب الكبار الذي لا يتطلب أي معدات متخصصة وصيانة قليلة جدا. نحن لشرح تشريح وحضانة للشبكية الأرانب والجسيمات بوساطة نقل الجينات من البلازميدات GFP ترميز أو مجموعة متنوعة من العلامات التحت خلوية إلى خلايا الشبكية العقدة. شبكيات الأرانب مثقف يمكن أن تبقى على قيد الحياة بهذه الطريقة لمدة تصل إلى 6 أيام مع تغييرات قليلة جدا من بنية تشريحية أو التشكل العام للدول ، فرادى وخلايا العقدة عديم الاستطالات.

Watch this Scientific Journal Video about Organotypic Culture of Adult Rabbit Retina at JoVE.com

Organotypic Culture of Adult Rabbit Retina

هذا المقال يوضح تشريح وحضانة للشبكية الأرانب والجسيمات بوساطة نقل الجينات من البلازميدات GFP ترميز أو مجموعة متنوعة من العلامات التحت خلوية إلى خلايا الشبكية العقدة.

عضوي النمط من الثقافة الشبكية أرنب الكبار

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

organotypic-culture-of-adult-rabbit-retina

Research

JoVE Journal

Biology

11.9K Views.  MGH - Massachusetts General Hospital. هذا المقال يوضح تشريح وحضانة للشبكية الأرانب والجسيمات بوساطة نقل الجينات من البلازميدات GFP ترميز أو مجموعة متنوعة من العلامات التحت خلوية إلى خلايا الشبكية العقدة.

Video: Organotypic Culture of Adult Rabbit Retina

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

إعداد شريحة الأفقي للشبكية

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

عضوي النمط من الثقافة شريحة أدمغة الفئران E18

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

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 ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

في الجسم الحي مثل الفئران عضوي النمط الثقافة Wholemount الشبكية

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

الكبار والعضلات والهيكل العظمي الجنينية Microexplant الثقافة وعزل خلايا جذعية عضلية هيكلية

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

العزلة وثقافة الكبار الخلايا الجذعية الظهارية من الجلد البشري

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

في الجسم الحي من Electroporation Morpholinos في شبكية العين اسماك الزرد الكبار

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

العزلة وثقافة فردية وMyofibers زنزاناتهم القمر الصناعي من العضلات الهيكلية الكبار

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

العزلة والثقافة، وتوصيف وظيفي من الكبار ماوس Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

العزلة وثقافة الأسنان طلائي الخلايا الجذعية من الكبار ماوس القاطعة

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...