تحريض DTH بالتبني الرجاء راجع المقالة إن الرب : <a href="/index/Details.stp?ID=325">الاستقراء والرصد من فرط الحساسية المتأخرة نوع بالتبني في الفئران</a> كريستين Beeton جورج ك. شاندي قسم الفسيولوجيا والفيزياء الحيوية في جامعة كاليفورنيا في ايرفين تجارب J. متصور ، العدد 8 هنا ، ونحن قد عدلت هذا البروتوكول باستخدام المستضد (Ovalbumin) مترافق الى تكساس الأحمر في نسبة 1:1 مع مستضد غير المسماة للسماح التصور من الخلايا البلعمية مستضد تقديم. الأذن الأنسجة الحصاد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الموت ببطء والفئران عند نقطة في الوقت المطلوب عن طريق الإجراءات المحددة في البروتوكول الخاص الحيوانية المعتمدة. هنا ، فإننا نستخدم جرعة زائدة من المخدر ، isoflurane. يتم ذلك في حاوية مغلقة تقع داخل غطاء جيد التهوية. ضمان للحيوان ميت إما قطع الرأس من خلال فتح التجويف الصدري وقطع القلب. </li><li style=";text-align:right;direction:rtl"> باستخدام مقص كبير إزالة قطع الأذن مع واحد ، على مقربة من جسم الحيوان قدر الإمكان (الشكل 1). <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> الشكل 1. إزالة الأذن الفئران </li><li style=";text-align:right;direction:rtl"> مكان الأذن في أنبوب مخروطي 15 مل تحتوي على ~ 10 مل من 1X PBS الجليد الباردة أو RPMI 1640. الحفاظ على عينات الأنسجة على الجليد حتى تكون على استعداد لتحضير لتصوير الأنسجة. </li><li style=";text-align:right;direction:rtl"> صورة الأنسجة في أقرب وقت ممكن ، ونحن نجد ، على الرغم من أن 1 إلى 2 ساعة على الجليد ليس له تأثير ملحوظ على حركية الخلية في الأنسجة عند مقارنة النسيج المقطوع والتقط على الفور. </li></ol> التحضير لتصوير الأنسجة أولا إزالة طبقة البشرة والجلد * ملاحظة : يجب أن تبقى طبقة الجلد العميقة سليمة. إذا كان القيام به بشكل صحيح سوف الأوعية الدموية الكبيرة لا تزال سليمة وسوف لا يتعرض الغضروف في الأذن يكون. هذا هو أسلوب صعب ، وخصوصا عندما لا يوجد التهاب (شروط المراقبة) وانه من المفيد استخدام مجهر تشريح. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان كل من الأنسجة وسائل الإعلام من الأنبوب المخروطية في صحن 10 سم زراعة الأنسجة. </li><li style=";text-align:right;direction:rtl"> مع الاستمرار على أيدي القفاز الأذن الفئران بلطف على طرف الأذن (البعيدة) مع الجانب الظهري من الأذن مواجهة (الشكل 2). <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> الشكل 2. تقليم الفراء قبالة الأذن </li><li style=";text-align:right;direction:rtl"> مع إبهامك على أعلى الأذن ، والنسيج ثابتة أثناء استخدام غيض من دومون مغلقة # 5 المقص لفصل الأدمة العميقة من الأدمة. بدلا من ذلك ، إذا كان على حافة الأذن لديها فائض من الجلد / الأدمة العميقة ، يمكن اغتنامها هذا مع ملقط وسحبت بلطف لفصل هذه الأنسجة من تحت (الشكل 3). <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> الشكل 3. إزالة الأدمة العليا </li><li style=";text-align:right;direction:rtl"> مرة واحدة وقد تم فصل منطقة صغيرة من الجلد من الأنسجة الكامنة ، كرر الخطوات بدلا من الانفصال باستخدام مقص لقطع بين طبقات الجلد وملقط لإزالة بلطف وتقشر الجلد فكها. </li><li style=";text-align:right;direction:rtl"> تكشف النقاب عن 5 × 5 ملم أو مساحة أكبر من أنسجة الأذن التي لا تقل عن 3 ملم من موقع الأولي لحقن المستضد (الشكل 4). <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> الشكل 4. الأنسجة تتعرض للتصوير </li></ol> II. تأمين الأنسجة إلى مرحلة التصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع من البلاستيك لتغطية زلة حجم أكبر قليلا من نسيج المعدة. </li><li style=";text-align:right;direction:rtl"> انتشار طبقة رقيقة من نسيج Vetbond 3M ™ آمنة الغراء عبر الشريحة. </li><li style=";text-align:right;direction:rtl"> فهم ركن من نسيج المعدة (حيث كنت من غير المحتمل أن الصورة) ، وترفع من وسائل الإعلام ، وربت الجانب السفلي من الأنسجة على الورق أو عدسة كيم مسح وسائل الإعلام لإزالة الزائدة. </li><li style=";text-align:right;direction:rtl"> لمس حافة الأنسجة لتغطية الشريحة الغراء (ويشفي Vetbond فورا على النسيج الرطب). تمتد برفق الأنسجة شقة ، مع الطرف المقابل هو آخر من لمس الشريحة (الشكل 5) <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> الشكل 5. جبل نسيج لتغطية زلة </li><li style=";text-align:right;direction:rtl"> علاج ™ Vetbond بواسطة قلب النسيج عينة / الانزلاق والغمس في خزان من وسائل الاعلام ان هذه الشريحة والنسيج وجهه لأسفل وتقريبا بزاوية 45 درجة. عقد النسيج رأسا على عقب في زاوية يساعد على منع الغراء الزائد من على سطح علاج الأنسجة المكشوفة. والغراء على سطح النسيج كتلة ليزر (الشكل 6). <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> الشكل 6. شفاء الأنسجة vetbond حمض الغلوتاميكه عن طريق غمس في وسائل الإعلام ملاحظة : خطوات 02-05 مايو يأخذ القليل من الممارسة لاتقان. حاول ممارسة القطع مع تجاهل أو الأنسجة الزائدة قبل محاولة للمرة الأولى. </li><li style=";text-align:right;direction:rtl"> ربت على كمية صغيرة من السيليكون الشحوم على الجزء السفلي من الشريحة. </li><li style=";text-align:right;direction:rtl"> ضع الشريحة في غرفة الارواء. تأكد من أن وسائل الإعلام نضح تتدفق ، و 37 درجة مئوية ، والاوكسيجين قبل ان يضيف الأنسجة. اضغط برفق لأسفل على حواف الشريحة صنع ختم الشحوم مع السيليكون بين الجزء السفلي من الغرفة ، ونضح زلة غطاء من البلاستيك. </li></ol> III. ثنائي الفوتون التصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام الضوء الساطع مجال المنقولة ، والتركيز على الجزء العلوي من الأنسجة. إيقاف كل الاضواء. </li><li style=";text-align:right;direction:rtl"> بدوره على الليزر ، وهذه الفرق على النحو المطلوب من قبل النظام الخاص مجهر متعدد الفوتون. </li><li style=";text-align:right;direction:rtl"> الجيل الثاني التوافقي (SHG) في البروتينات الليفية عالية أمر (الكولاجين والميوسين) تنتج إشارة في الطول الموجي النصف من الليزر الإثارة. نستخدم الإثارة في 900 نانومتر ، مما أدى إلى SHG عند 450 نانومتر التي يمكن تصور استخدام &quot;الزرقاء&quot; قناة المجهر. SHG جيل هو غير الخطية (ثنائي الفوتون) عملية ، ويوفر بالتالي البصرية &quot;sectioning&quot; تأثير مماثل لذلك من الإثارة اثنين من الفوتون مضان. كفاءة ذروة SHG يحدث عندما يتم ييفات عمودي على الليزر ، في حين أن الألياف تعمل في موازاة ذلك لا يمكن تصور 1 </li><li style=";text-align:right;direction:rtl"> موقع التصوير في منطقة جيدة مع الكثير من الخلايا ، وتعيين منطقتك اقتناء مثل هذه الغالبية من الخلايا الموجودة في المركز وبدء التصوير. ملاحظة : في ردود الفعل المباشرة الى البيوت ، وكثيرا ما توجد الخلايا التائية والخلايا الأخرى تتسرب إلى التركز معا بينما مناطق أخرى خالية من التسلل الى الخلايا 2. لذلك ، قد يستغرق بعض الوقت في البحث في النسيج للعثور على أفضل صورة للمنطقة. </li><li style=";text-align:right;direction:rtl"> الاختيار لبقاء الخلية من خلال تقييم سرعة الخلية والاقطاب خلال التصوير. استخدام قوة أقل الليزر الذي يوفر جودة الصورة مقبولة لضمان الحد الأدنى من الصور والضرر. </li><li style=";text-align:right;direction:rtl"> مبادلة في إعداد أنسجة جديدة حسب الحاجة. </li></ol>

<ol>
	<li>Koo, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+the+voltage-gated+potassium+channel+Kv1.3+inhibits+immune+responses+in+vivo.">Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.</a> J Immunol. 158, 5120-5128 (1997).</li><li>Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+activation+and+T+lymphocyte+infiltration+in+allergen-induced+late+phase+skin+reactions+and+classical+delayed-type+hypersensitivity.">Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity.</a> J Immunol. 147, 816-822 (1991).</li><li>Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoaggressive+effector+T+cells+in+the+course+of+experimental+autoimmune+encephalomyelitis+visualized+in+the+light+of+two-photon+microscopy.">Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy.</a> J Neuroimmunol. 191, 86-97 (2007).</li><li>Kawakami, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+effector+cell+trafficking+and+autoantigen+recognition+within+the+unfolding+autoimmune+encephalomyelitis+lesion.">Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.</a> J Exp Med. 201, 1805-1814 (2005).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Matheu, M. P. B., A, C. G. a. r. c. i. a., Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Imaging+of+Effector/Memory+T+Cells+During+DTH+and+Suppression+by+Kv1.3+Channel+Block.">In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block.</a> Immunity. , (2008).</li></ol>

imaging effector memory t cells in the ear after induction of adoptive dth

تأخر فرط نوع (DTH) هو رد الفعل المناعي الذي اللاعبين الرئيسيين هم CCR7 -- المستجيب / اللمفاويات التائية الذاكرة. هنا ، علينا أن نظهر وسيلة لحفز التقدم وتسجيل ردود فعل من البيوت في الأذن الفئران. اعقب ذلك عرض لإعداد الأنسجة الأذن الفئران لتصوير اثنين من الفوتون CCR7 -- المستجيب / الذاكرة استجابة الخلايا التائية. هو أحد البيوت التي يسببها بالتبني من قبل حقن داخل الصفاق من GFP المسمى البويضات محددة CCR7 -- المستجيب / الذاكرة خط الخلايا التائية (Beeton ، C J. التجارب متصور ، العدد 8). ثم يتم السماح للتوازن الخلايا في الفئران لمدة 48 ساعة قبل التحدي عن طريق حقن أذن واحدة مع المياه المالحة (مراقبة الأذن) ، والآخر مع مزيج من مترافق 01:01 البويضات والبويضات الى تكساس الأحمر (OVA - TR) للسماح التصور الخلايا مستضد تقديم المقيمين. نحن تصف طريقة لإعداد الأنسجة مفيدة لتصوير حركية من خلايا داخل طبقة الجلد العميقة خلال استجابة مناعية ، بالاشتراك مع التصور من ألياف الكولاجين التي التوافقي الجيل الثاني. يتم قطع نسيج الأذن إلى 5 مم X المربعات 5 (أكبر قليلا هو أفضل) والتي شنت على غطاء بلاستيكي باستخدام قسائم Vetbond ™ ، والتي يتم تأمينها ثم استخدام الشحوم سيليكون في غرفة التصوير وsuperfused بواسطة الأكسجين فقاعات نسيج الثقافة في المتوسط ​​37 درجة مئوية .

Watch this Scientific Journal Video about Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH at JoVE.com

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

نحن هنا يبرهن على وجود طريقة لحمل وتسجيل التقدم المحرز في رد فعل متأخر (DTH) نوع فرط في الأذن الفئران. اعقب ذلك عرض لإعداد الأنسجة الأذن الفئران لتصوير ثنائي الفوتون من المستجيب / الذاكرة استجابة الخلايا التائية.

التصوير T الذاكرة المستجيب الخلايا في الأذن بعد تحريض DTH بالتبني

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

imaging-effector-memory-t-cells-ear-after-induction-adoptive

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K Views.  University of California, Irvine (UCI). نحن هنا يبرهن على وجود طريقة لحمل وتسجيل التقدم المحرز في رد فعل متأخر (DTH) نوع فرط في الأذن الفئران. اعقب ذلك عرض لإعداد الأنسجة الأذن الفئران لتصوير ثنائي الفوتون من المستجيب / الذاكرة استجابة الخلايا التائية.

Video: Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

الاستقراء والرصد من أحدث نوع فرط الحساسية المتأخرة (DTH) في الفئران

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

الاستقراء والرصد من فرط الحساسية المتأخرة نوع بالتبني في الفئران

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

الابتدائية الثقافة وElectroporation البلازميد الجهاز الفئران كورتي.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

مولد الرمع الفحص : خلايا منتسب

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

يعيش التصوير من النتوء خلية من البشرة من اسماك الزرد النامية

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

تصوير مستقبلات الاستروجين في الشرايين α بيال فأر باستخدام مجهر رقمي مناعي

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

التصوير مضان مع نانومتر دقة واحدة (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

التصوير المحلية الكالسيوم<sup&gt; 2+</sup&gt; الإشارات في خلايا الثدييات مثقف

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

عالية الدقة التصوير الوقت الفاصل بين وتحليل الآلي للأنيبيب حيوية في المعيشة السري الإنسان خلايا الوريد غشائي

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...