وأيد هذا العمل من قبل جمعية الألمانية للبحوث (DFG؛ KL 1415/3-2 وKL 1415/4-2).

1. إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد أطباق ثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الكولاجين النوع الرابع O / N عند 4 ° C وحلها في 0.1٪ حامض الخليك العقيمة. حجم لاستخدام يعتمد على نوع وعدد من الأطباق التي كانت الخلايا ليكون المصنف. استخدام 2 ميكروغرام / سم 2. </li><li style=";text-align:right;direction:rtl"> احتضان الأطباق على الأقل لمدة 1 ساعة على RT ويغسل مرتين مع الماء المقطر (A. tridest). </li><li style=";text-align:right;direction:rtl"> السماح للأطباق لتجف بشكل صحيح. </li></ol></li><li style=";text-align:right;direction:rtl"> مكملات من المتوسطة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> زيادة مستوى الجلوكوز يصل إلى 4.5 جرام / لتر وذلك بإضافة 1.75 ز الجلوكوز إلى 500 مل المتوسطة. لضبط المتوسطة إلى 600 mosmol، إضافة 100 ملي مول كلوريد الصوديوم واليوريا 100 ملم، لزيادة الأسمولية بنسبة 200 mosmol و 100 mosmol، على التوالي. </li><li style=";text-align:right;direction:rtl"> إضافة 1٪ الأحماض الأمينية غير الأساسية، 1٪ ultroser، 500 DBcAMP ميكرومتر، 20 U / مل نيستاتين و 0.25 ميكروغرام / مل جنتاميسين (1:200 التخفيف من محلول المخزون مع تركيز 50 ميكروغرام / مل) إلى 600 mosmol DMEM المتوسطة طازجة في اليوم من التحضير. </li></ol></لى&gt; <li style=";text-align:right;direction:rtl"> حل انزيم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 1 ملغ / مل هيالورونيداز و 2.2 ملغ / مل كولاجيناز إلى DPBS المحتوية على 0.25 ميكروغرام / مل جنتاميسين والنيستاتين. إعداد 1 مل من هذا الحل انزيم لهضم 2 الكلى للنخاع الداخلية. يجب تعقيم الحل عن طريق الترشيح ويتم تخزينها في 50 مل أنابيب فالكون. </li></ol></li></ol> 2. زراعة النخاع الابتدائية الجرذ الداخلية جمع مجاري الهواء (IMCD) خلايا جميع إجراءات التعامل مع الحيوانات هي التي يتعين الاضطلاع بها في الامتثال للتشريعات المحلية والاتحادية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الفئران في CO 2 (في مربع مشبعة CO 2) أو isoflurane و(U-400 وحدة التخدير، Univentor المحدودة، مالطة؛ تدفق الهواء 350-400 مل / دقيقة مع 2-2.5٪ isoflurane و). قطع رأس الحيوانات لوقف نزيف الذي يسمح الكشف الدقيق للحدود بين بيضاء الداخلية والنخاع الخارجي أكثر قتامة. وبالإضافة إلى ذلك، النزيف يقلل من عدد كرات الدم الحمراء المعزولة. إزالة الكلى وغسلها في DPBS العقيمة تستكمل مع الجنتاميسين والنيستاتين (انظر أعلاه) على الجليد. جميع خطوات أخرى هي التي يتعين الاضطلاع بها تحت ظروف معقمة مقاعد البدلاء النظيفة. </li><li style=";text-align:right;direction:rtl"> نقل الكلى من حل DPBS على ضغط معقمة لإزالة السائل الزائد. إزالة الكبسولة الكلى الدهنية مع ملقط معقم، ومقص. </li><li style=";text-align:right;direction:rtl"> وبعد أن زال الكلى ثابتة في ضغط، وقطع قليلا بجانب المحور الطولي للجانب الخارجي (القشرية). تأكد من عدم قطع طريق تماما، ولكن ترك الأمر في قطعة واحدة، مرتبطة في الحوض الكلوي. </li><li style=";text-align:right;direction:rtl"> مع مقص منحني تشريح بعناية للنخاع الداخلية بيضاء، والتي تحيط بها النخاع الخارجي متلألئ روز. جمعها في DPBS، التي تحتوي على جنتاميسين والنيستاتين في 60 ملم صحن الثقافة على الجليد. </li><li style=";text-align:right;direction:rtl"> مرة واحدة يتم عزل جميع للنخاع والتي تم جمعها على الجليد، والحد من حجم DPBS إلى مستوى حيث يتم تغطيتها فقط مع العازلة. استخدام razorblade معقمة لقطع للنخاع إلى مكعبات من 5مم 3. </li><li style=";text-align:right;direction:rtl"> تذوب وجولة على نهاية حادة من العقيمة باستور ماصة عن طريق إمساكه لفترة وجيزة في لهب. تأكد يتم تبريد ماصة قبل المتابعة. </li><li style=";text-align:right;direction:rtl"> نقل تعليق الأنسجة إلى 50 مل أنابيب فالكون يحتوي الطازجة حل انزيم العقيمة مع هيالورونيداز وكولاجيناز (انظر 1.3.1) باستخدام مدورة ماصة باستير. إغلاق الغطاء بإحكام واحتضان عند 37 درجة مئوية في إطار التحريض المستمر (220 دورة في الدقيقة) في حمام المياه العادية الطاولة ل1،5-2 ساعة. </li><li style=";text-align:right;direction:rtl"> لسحن لتعليق، ماصة الخليط صعودا وهبوطا عدة مرات مع الجولة ماصة باستير (انظر أعلاه) حتى يصبح عكر متجانس تعليق. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي تعليق لمدة 5 دقائق في 300 XG و 16 درجة مئوية. تجاهل طاف، resuspend الكرية جيدا في DPBS، التي تحتوي على جنتاميسين والنيستاتين وأجهزة الطرد المركزي مرة أخرى. كرر هذه الخطوة غسل مرة واحدة. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا في المتوسط ​​تستكمل بالكامل والبذور لهم في جأطباق ulture و / أو لوحات جيدا عيار الصغرى. وإعداد 2 للنخاع (1 الحيوان) تسفر عن خلايا كافية لواحد صحن الثقافة 60 ملم. </li><li style=";text-align:right;direction:rtl"> بعد 24 ساعة، وغسل الخلايا مرتين مع 600 mosmol DMEM وإضافة المتوسطة الطازجة. خلال الأسبوع المقبل وينبغي غسل الخلايا 2-3 مرات (الشكل 1). بعد 6-8 أيام البذر، وخلايا IMCD يمكن استخدامها لإجراء التجارب. تذكر لاحتضان لهم في وسيط وبدون DBcAMP ونيستاتين لمدة 24 ساعة قبل بدء التجربة. وإلا AQP2 سوف يكون موجودا حصرا في غشاء البلازما. </li></ol>

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

نقدم بروتوكول مفصلة لإعداد وزراعة الخلايا IMCD الفئران الأولية. النهج غلة تصل إلى 21 سم 2 من الخلايا من الفئران واحدة 20. التجربة يتطلب معيار معدات زراعة الخلايا ويمكن أن يقوم بها شخص واحد في غضون ما يقرب من 6 ساعة. ولذلك، فإن هذا النهج هو مناسبة كوسيلة المختبر ?...

A correction was made to <a href="http://www.jove.com/video/50366">Culturing Primary Rat Inner Medullary Collecting Duct Cells</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Enno Klussmann instead of: Enno Klussman

A correction to author list has been made for article Culturing Primary Rat Inner Medullary Collecting Duct Cells.

Erratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells

في الكلى جمع الخلايا الرئيسية لاصق، أرجينين-فاسوبريسين (AVP) تسيطر على استيعاب المياه من خلال تحفيز الإدراج من الماء قناة aquaporin-2 (AQP2) في غشاء البلازما. AVP بربط البروتين يقترن G فاسوبريسين نوع 2 مستقبلات (V2R) تحفيز محلقة أدينيليل وبالتالي تشكيل المخيم. بدء هذه السلسلة يشير يؤدي إلى تنشيط بروتين كيناز ألف (PKA). PKA phosphorylates AQP2 في سيرين 256 (S256)، والذي هو المحرك الرئيسي لإعادة توزيعها في الفترة من حويصلات داخل الخلايا في غشاء البلازما. إدراج غشاء يسهل استيعاب المياه على طول الانحدار ناضح وغرامة الإيقاعات الجسم التوازن المياه. ويرتبط التقلبات من استيعاب المياه AVP بوساطة، وذلك بسبب الانحرافات من إفراز AVP أو أسباب إشارات AVP تنشيط أو يعانون من أمراض حادة. انخفضت استيعاب المياه التي تسببها طفرات من V2R أو AQP2 يؤدي إلى مرض السكري الكاذب كلوي، في حين أن ELEVويرتبط انبعاث العوادم بلازما الدم مستوى AVP مع استيعاب المفرط للمياه في أمراض القلب والأوعية الدموية مثل قصور القلب المزمن ومتلازمة غير لائق المضاد لإدرار البول إفراز هرمون (SIADH). أهمية استيعاب المياه AVP بوساطة لا ينبع فقط من آثاره في المرض. وإزفاء AVP التي يسببها من الحويصلات AQP2 الحاملة لوالانصهار مع غشاء البلازما يمثل عملية exocytic بدقة مخيم معتمدة، وهو حاليا غير مفهومة جيدا. أمثلة أخرى لإيماس التي تعتمد على المخيم إفراز الرينين في الكلى وH + إفراز في المعدة. ولذلك، توضيح من الآليات الجزيئية التي تنظم AQP2-إزفاء في الخلايا الرئيسية الكلوي لا يساعد فقط على فهم العمليات الجزيئية مماثلة في أنواع الخلايا الأخرى ولكن أيضا قد يمهد الطريق لعلاجات جديدة لعلاج الأمراض المرتبطة اضطرابات من استيعاب المياه AVP بوساطة . Elucidating آليات السيطرة AQP2 يتطلب جمع القناة نماذج الخلية الرئيسية. لهذا عدد من مختلف الثدييات خطوط الخلايا الكلى متوفرة. ومع ذلك، هذه النماذج لديها العديد من السلبيات. في كثير من النظم خلية مستوى البروتين من AQP2 منخفضة (الجدول 1؛ M1، HEK293، COS-7، MDCK، LLC-PK1) 1-10، والبعض الآخر ectopically overexpress الإنسان (MCD4، WT10) 11،12 أو الفئران (CD8) 13 AQP2. في MCD4 وCOS-7 الخلايا لا يتم التعبير عن مستقبلات V2. على حد علمنا ليس هناك خط خلية دائمة كنموذج المتاحة، والتي هي مستمدة من النخاعية الداخلية الكلى جمع القناة، أن جزءا من الكلى حيث AQP2 التعبير هو الأكثر وفرة، ولكن من القشرية جمع القناة 1،11،13-16 . نماذج الخلية مثل هذه هي على سبيل المثال تستخدم على نطاق واسع خلد mpkCCD (مثلا 17) أو mTERT-CCD 14 خطوط الخلايا التي أنشئت مؤخرا. كلا الخطين الخلية التطور الطبيعي صريحة AQP2 ولكن منذ V2R أنها مشتقة منالقشرية القناة جمع الأرجح تحتوي على بروتيوم مختلفة مقارنة النخاعية الداخلية جمع مجاري الهواء (IMCD) الخلايا. يجب عليهم على سبيل المثال التعبير عن مختلف نا + أنظمة النقل. هنا، نقدم بروتوكول لثقافة الخلايا IMCD الابتدائية تعبر عن V2R وAQP2 التطور الطبيعي. هذا النموذج، وبالتالي، يمثل أكثر عن كثب الوضع الفسيولوجية في القناة جمع الكلوي. كما يتطلب إنشاء ثقافة فقط المعدات المختبرية القياسية مختبرات أخرى يمكن اعتماد هذا النهج بسهولة.

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culturing primary rat inner medullary collecting duct cells

أرجينين-فاسوبريسين (AVP) يسهل إعادة امتصاص الماء بواسطة الكلى جمع الخلايا الرئيسية لاصق، وبالتالي غرامة الإيقاعات الجسم توازن الماء. AVP تربط لمستقبلات V2 فاسوبريسين (V2R) على سطح الخلايا وبالتالي يدفع توليف المخيم. هذا يحفز عمليات الإشارات الخلوية مما يؤدي إلى تغييرات في الفسفرة من قناة المياه aquaporin-2 (AQP2). بروتين كيناز A phoshorylates AQP2 وبالتالي يتسبب في إزفاء من AQP2 من حويصلات داخل الخلايا في غشاء البلازما تسهيل استيعاب الماء من البول الابتدائي. الانحرافات الإفراج AVP من إشارات النخامية أو AVP تنشيط في الخلايا الرئيسية يمكن أن يسبب مرض السكري الكاذب المركزي أو كلوي، على التوالي؛ يرتبط على الدم مرتفعة البلازما مستوى AVP مع أمراض القلب والأوعية الدموية مثل قصور القلب المزمن ومتلازمة غير لائق المضاد لإدرار البول إفراز الهرمون. هنا، نقدم بروتوكول للcultivatiعلى من الفئران النخاعية الداخلية الابتدائي جمع مجاري الهواء (IMCD) الخلايا والتي تعبر V2R وAQP2 التطور الطبيعي. الخلايا هي مناسبة لتوضيح الآليات الجزيئية الكامنة وراء سيطرة AQP2 وبالتالي لاكتشاف الأهداف المخدرات رواية لعلاج الأمراض المرتبطة التقلبات من استيعاب المياه AVP بوساطة. ويتم الحصول على الخلايا IMCD من الفئران للنخاع الداخلية الكلوي وتستخدم للتجارب بعد ستة إلى ثمانية أيام البذر. يمكن تربيتها خلايا IMCD في الأطباق العادية خلية ثقافة والقوارير واللوحات عيار الصغرى من الأشكال المختلفة، فإن الإجراء يتطلب بضع ساعات فقط، وغير مناسبة للمختبرات زراعة الخلايا القياسية.

فإن زراعة ناجحة لخلايا الفئران IMCD الأولية يؤدي إلى أحادي الطبقة متموجة بعد 6-8 أيام البذر (الشكل 2). لكل 60 مم طبق الثقافة هناك ما يقرب من 6 × 10 6 خلايا. الخلايا التمسك بإحكام إلى أطباق ثقافة، كما كانت مغلفة هذه مع الكولاجين النوع الرابع، وهو أحد مكونات الغشا...

Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Culturing Primary Rat Inner Medullary Collecting Duct Cells

أرجينين-فاسوبريسين (AVP) تسيطر على صقل الجسم توازن المياه من خلال تسهيل استيعاب المياه بواسطة الخلايا الرئيسية الكلوي. هنا، نقدم بروتوكول لزراعة النخاع الفئران الداخلية الابتدائي جمع خلايا القناة مناسبة لتوضيح الآليات الجزيئية الكامنة وراء إعادة امتصاص الماء AVP بوساطة.

زراعة النخاع الابتدائية الجرذ الداخلية جمع خلايا القناة

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

culturing-primary-rat-inner-medullary-collecting-duct-cells

Research

JoVE Journal

Biology

14.7K Views.  Max-Delbrück-Center for Molecular Medicine. أرجينين-فاسوبريسين (AVP) تسيطر على صقل الجسم توازن المياه من خلال تسهيل استيعاب المياه بواسطة الخلايا الرئيسية الكلوي. هنا، نقدم بروتوكول لزراعة النخاع الفئران الداخلية الابتدائي جمع خلايا القناة مناسبة لتوضيح الآليات الجزيئية الكامنة وراء إعادة امتصاص الماء AV...

Video: Culturing Primary Rat Inner Medullary Collecting Duct Cells

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

جمع وقياس وسطاء Nociceptive والتهاب في الجروح الجراحية

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

بروتوكول للالخلايا العصبية التثقيف تعاطفية من العقد الجرذ عنق الرحم فخمة (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

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 ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

عزل الخلايا القاعدية والخلايا تحت المخاطية قناة الغدة من القصبة الهوائية ماوس

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

العزلة والثقافة الأساسي في الخلايا الكبدية الجرذ

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

عزل الخلايا المناعية من الأورام الأولية

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

زراعة خلايا الإنسان الظهارية الأنف في واجهة الهواء السائل

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

منهج في التثقيف الجنينية<em&gt; C. ايليجانس</em&gt; خلايا

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

الثقافة الأولية من خلايا الفئران البطاني وفحوصات لوظائف ستيرويدوجينيك

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

عزل خلايا كبد الفئران الأولية مع التحكم في التروية متعدد المعلمات

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...