ونحن ممتنون لايواتا كين (OSI الصيدلة ، نيويورك ، الولايات المتحدة) عن الكواشف وفريد ​​ميلر (باربرا آن كارمانوس للسرطان Intitute ، ديترويت ، الولايات المتحدة الأمريكية) لخطوط الخلية.

1. إعداد الحل methocel (500 مل) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأوتوكلاف 6 غرام من methylcellulose في زجاجة 500 مل تحتوي على مغناطيسي. </li><li style=";text-align:right;direction:rtl"> حل methylcellulose في 250 مل من دون DMEM/F12 محمى المصل (60 درجة مئوية) لمدة 20 دقيقة. </li><li style=";text-align:right;direction:rtl"> إضافة 250 مل من DMEM/F12 (RT) تحتوي على 10 ٪ مصل الحصان. مزيج بين عشية وضحاها (4 درجة مئوية). </li><li style=";text-align:right;direction:rtl"> الحل واضح عن طريق الطرد المركزي (5000g ، 2H ، 4 درجات مئوية). </li><li style=";text-align:right;direction:rtl"> اتخاذ حل واضح للغاية للزج أنبوب جديد (حوالي 90-95 ٪ من محلول المخزون). </li><li style=";text-align:right;direction:rtl"> تخزين عند 4 درجات مئوية حتى الاستخدام. </li></ol> 2. ثقافة كروي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يعد حل methocel 20 ٪ (10 مل و 40 methocel المتوسطة النمو مل). </li><li style=";text-align:right;direction:rtl"> غسل الخلايا مرتين مع برنامج تلفزيوني ، إضافة التربسين 0.1 ٪ والخلايا احتضان عند 37 درجة مئوية </li><li style=";text-align:right;direction:rtl"> Resuspend الخلايا المتوسطة في النمو وعدد الخلايا. </li><li style=";text-align:right;direction:rtl"> إعداد تعليق من الخلايا 000 مل في 10 في methocel 20 ٪. </li><li style=";text-align:right;direction:rtl"> إضافة تعليق الخلية إلى أسفل ق 96 جولة جيدالوحات uspension (100 ميكروليتر لكل بئر). </li><li style=";text-align:right;direction:rtl"> اليوم التالي ، والتحقق من تكوين كروي في لوحات تعليق. الأجسام الشبه الكروية سوف يكون جاهزا للاستخدام بعد 1-3 أيام. </li></ol> 3. إعداد الكولاجين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد على الجليد حل الكولاجين مل 8 ، 1 مل 10X PBS ، 1 مل هيدروكسيد الصوديوم 0.1 ن 0.1 و 3 قطرات N حمض الهيدروكلوريك. معرفة ما اذا كان الرقم الهيدروجيني هو 7.4 من خلال الكشف عن بعض الحل على شرائط مؤشر الرقم الهيدروجيني. ضبط درجة الحموضة إذا كان خارج النطاق. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكروليتر من محلول الكولاجين تحييدها في الآبار من 96 لوحة جيدا </li><li style=";text-align:right;direction:rtl"> احتضان عند 37 درجة مئوية حتى توطد الكولاجين (90 دقيقة). </li><li style=";text-align:right;direction:rtl"> إعداد methocel - الكولاجين يجند الحلول على الجليد : ليجند كل جزء 1 ماصة من حل الكولاجين. إضافة 20 مل من الكولاجين في نانوغرام TGF - β. بعد التخفيف مع methocel ونشر أكثر من طبقات مختلفة ، وسيكون التركيز النهائي للβ - TGF تكون 5 نانوغرام / مل ميكس بواسطة vortexing. إضافة 1 جزء من الحل ، methocel. مزيج من قبل vortexing. </li></ol> 4. تضمين كرويقرع <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان غيض 200 ميكروليتر الذي فقد ما يقرب من 5 ملم من الحافة قطع ، على ماصة 200 ميكروليتر. مص واحدة كروي مع ماصة والاستغناء بعناية المتوسطة في لوحة فارغة. اذا كان يراقب كروي يبقى داخل الحافة. وكروي مرئيا باعتبارها نقطة بيضاء. من دون الافراج عن المكبس ، تمتص 100 ميكروليتر الكولاجين methocel الخليط وصرف كروي في الخليط الكولاجين في الكولاجين 96 المغلفة جيدا. القيام بذلك لمدة 12 الأجسام الشبه الكروية لكل حالة. </li><li style=";text-align:right;direction:rtl"> اسمحوا الكولاجين ترسيخ ما لا يقل عن 30 دقيقة عند 37 درجة مئوية </li><li style=";text-align:right;direction:rtl"> إزالة فقاعات الهواء مع طرف 200 ميكروليتر. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر من المتوسط ​​1.6 ٪ مع المصل ومثبطات الذائبة في DMSO. لSB - 431542 إضافة 4 ميكرولتر من محلول المخزون (10 ملم) إلى 1 مل من المتوسط. بعد نشرها ، فإن التركيز النهائي للالمانع أن يكون 10 ميكرومتر. سوف TGF - β ومثبطات البقاء على مسار التجربة. التقاط صور للالأجسام الشبه الكروية تحت المجهر باستخدام التكبير 40X. </li><li style=";text-align:right;direction:rtl"> بعد 48h من التحفيز و / TGF - β أو مثبطات ، والتقاط صور من الأجسام الشبه الكروية تحت المجهر باستخدام التكبير 40X. </li><li style=";text-align:right;direction:rtl"> قياس مجال غزو الأجسام الشبه الكروية بعد 2 أيام ، وطرح منطقة الانطلاق. قياس مساحة كروي : افتح صورة من كروي في Adobe Photoshop Extended في (الشكل 2A) وحدد أداة التحديد السريع (الشكل 2B). ويتغير مؤشر الماوس إلى دائرة مع &quot;زائد&quot; (+) علامة (الشكل 2C). أثناء سحب المؤشر فوق كروي ، وسوف تعترف فوتوشوب حدود كروي. إذا تم تحديد منطقة خارج كروي ، لا يمكن استبعاد هذه المنطقة من الاختيار بالضغط على مفتاح Alt أو باستخدام أداة التحديد السلبية التي دلت على أ -- التوقيع في شريط الأدوات (). يتغير المؤشر إلى دائرة مع &quot;ناقص&quot; (--) توقع وعن طريق سحب المؤشر فوق المنطقة المحددة بشكل خاطئ ، تتم إزالة هذه المنطقة من الاختيار (الشكل 2D). إذا كان يمكن تحديد اللازمة ، ومناطق متعددة عن طريق الضغط على الشيخIFT الزر. لمزيد من العمل بدقة ، ويمكن تعديل حجم مؤشر الماوس عن طريق تغيير قطر الفرشاة في شريط الأدوات (2E الشكل). عندما يتم تحديد كروي كامل ، يتم حساب مساحة الاختيار عن طريق تحليل MEASURE في شريط القائمة أو عن طريق الضغط على Ctrl + Shift + M (الرقم 2F). سوف تظهر نافذة تسجيل القياس يظهر اسم الملف ومنطقة التحديد في بكسل ، وكذلك البيانات الأخرى (الشكل 2G). إذا تم قياس تحديدات متعددة ، السطر الأول يوضح مجموع جميع المجالات. وترد قياسات التحديدات الفردية في الأسطر أدناه. يمكن تصدير البيانات من جميع القياسات إلى ملف نصي ، وفتح في Excel. </li></ol> 5. ممثل النتائج : تحولت H - RAS مثالا للمقايسة مع كروي (M1) خلايا الثدي العادية MCF10A1 الظهارية ، MCF10AneoT (M2) وتعطى الخلايا المشتقة من MCF10CA1a M2 (M4) في الشكل رقم 3. أظهرت الخلايا M1 الغزو ضعيفة دونالتحفيز ، ولكن أفضل بكثير غزت على التحفيز التي كتبها β - TGF. في المقابل ، تحولت RAS - M1 - M2 المشتقة غزا بالفعل بكفاءة دون إضافة مؤثرات ، وزادت هذه الحظيرة 4-5 على TGF - β العلاج. أظهرت الخلايا M4 أقوى الغزو ، سواء مع وبدون TGF - β الإضافة. المقبل ، درسنا ما إذا كنا قد تتداخل مع TGF - β بفعل الغزو في هذا النموذج كروي باستخدام مثبطات الكيميائية. لهذا الغرض استخدمنا SB - 431542 ، والتماثلية ATP ومثبط انتقائي من النشاط كيناز من TβRI ، فضلا عن نوع IB activin مستقبلات (ActRIB) وactivin مستقبلات مثل كيناز (ALK) 7 12. كما هو متوقع ، SB - 431542 - منعت أكثر قدرة TGF β بفعل غزو M1 ، M2 و M4 الخلايا (الشكل رقم 4) ، مشيرا إلى أن غزو TGF - β المحرض هو TβRI - كيناز التابعة. بالإضافة إلى ذلك ، الغزو القاعدية من الأجسام الشبه الكروية كما أعاقت بشدة ، مما يوحي بأن غزو القاعدية أيضا ديمعلقة على β - TGF. <img alt="الشكل 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> مخطط تدفق الرقم 1. كروي للمقايسة و3D. الأولى ، هي مطلي الخلايا تحت ظروف غير ملتصقة في لوحات المعلقات على شكل حرف U. aggregrate الخلايا في الأجسام الشبه الكروية multcellular ويمكن أن تكون جزءا لا يتجزأ في مصفوفة الكولاجين. في هذه المصفوفة الكولاجين ، وأنهم قادرون على غزو. <img alt="الشكل 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> الشكل 2. الموسع الكمي لمنطقة الأجسام الشبه الكروية باستخدام أدوبي فوتوشوب. (أ) يتم فتح صورة كروي في أدوبي فوتوشوب الاختيار (ب) من Extendend أداة التحديد السريع (C) سحب المؤشر فوق كروي لتحديد مجال إزالة (D) كروي للمنطقة شملت استخدام wongly السلبية أداة التحديد السريع (E) تعديل حجم فرشاة أداة التحديد السريع على نحو أكثر دقة تحديد المنطقة (F) اختيارقياس الأمر لتسجيل (G) مثال لسجل القياس. <img alt="الشكل 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> الشكل 3. TGF - β بفعل غزو الأجسام الشبه الكروية من MCF10A1 خلدت عفويا (M1) (A) ، RAS - حولت MCF10AneoT (M2 (B) والمنتشر في MCF10CA1a المشتقة (M4) (C). M1 ، M2 و M4 الأجسام الشبه الكروية جزءا لا يتجزأ من في الكولاجين عولجوا من 5ng/ml β - TGF ل48h كما هو محدد. AC صور الممثل الذي اتخذ في يوم من تضمين وبعد ذلك بيومين. D الغزو كان كميا والفرق النسبي المنطقة يوم 2 تحت الصفر اليوم 0. يتم التعبير عن النتائج كما يعني ± SD مقتبس من 13. <img alt="الشكل 4A" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="الشكل 4B" src="/files/ftp_upload/3337/3337fig4b.jpg" /> هو تحول دون الرقم 4. غزو كروي بصل وTGF - β صنع بذ SB - 431542. كانت جزءا لا يتجزأ من الأجسام الشبه الكروية M1 ، M2 و M4 في الكولاجين وتعامل مع 5ng/ml β - TGF و / أو 10 ميكرومتر SB - 431542 كما هو محدد. صور الممثلة المأخوذة بعد 2 يوما (A). وكان كميا والفرق النسبي غزو المنطقة في يوم 2 تحت الصفر اليوم 0 (باء). يتم التعبير عن النتائج على النحو يعني ± SD مقتبس من 13.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> J. Pharmacol. Exp. Ther. 332, 821-821 (2010).</li></ol>

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أنشأنا نموذجا كروي التي MCF10A1 الخلايا الخبيثة ومشتقاته في غزو مصفوفة الكولاجين بطريقة TGF - β - تعتمد. أولا ، يتم تشكيل الأجسام الشبه الكروية في 96 بئرا لوحات تعليق الجولة القاع في وجود methylcellulose. بالطبع ، يمكن أن تستخدم أيضا على شكل حرف U بولي HEMA لوحات مغلفة لهذا الغرض. مطلو?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليق (اختياري) </td></tr><tr><td> ميثيل السليلوز </td><td> سيغما الدريخ </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> المتقدمة Biomatrix </td><td> 5005 - B </td><td></td></tr><tr><td> مؤشر الرقم الهيدروجيني الشرائط </td><td> ميرك </td><td> 9533 </td><td></td></tr><tr><td> 96 - جيدا تعليق لوحات </td><td> غرينر بيو واحد </td><td> 650 185 </td><td></td></tr><tr><td> 96 - TC التصاق جيد لوحات </td><td> غرينر بيو واحد </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF - β ومعارضة الأدوار في تطور سرطان الثدي عن طريق العمل كمركز الكابتة للورم في المرحلة الأولي ، ولكن تحفيز الغزو والانبثاث في 1،2 مرحلة لاحقة. علاوة على ذلك ، كثيرا ما هى overexpressed TGF - β في سرطان الثدي وتعبيرها يرتبط بسوء التشخيص والانبثاث 3،4. الآليات التي لا TGF - β يدفع الغزو مفهومة جيدا. TGF - β يتسبب ردوده الخلوية عبر النوع الثاني TGF - β (TβRII) والنوع الأول (TβRI) المستقبلات. عند تشكيل TGF - β المعقدة التي يسببها مغايرة التغصن ، TβRII phosphorylates في TβRI. وTβRI تنشيط يبدأ في الخلايا مما يشير المسار الكنسي Smads بواسطة مستقبلات phosphorylating (R - Smads) ، أي Smad2 وSmad3. تنشيط هذه R - Smads شكل مجمعات مغايرة التغصن مع Smad4 والتي تتراكم في نواة وتنظيم النسخ للجينات الهدف 5. بالإضافة إلى د سابقامسار Smad escribed ، والنتائج في تنشيط مستقبلات تفعيل عدة مسارات أخرى غير Smad يشير ، على سبيل المثال Mitogen المنشط بروتين كيناز (MAPK) 6 مسارات. لدراسة دور β - TGF في مراحل مختلفة من سرطان الثدي ، والتي قطعناها على أنفسنا استخدام نظام MCF10A الخلية. حولت H - RAS هذا النظام يتألف من خلايا خلدت عفويا MCF10A1 الثدي (M1) الظهارية 7 ، M1 - المشتقة MCF10AneoT (M2) ، والتي تنتج آفات مرحلة ما قبل 8 في الفئران ، وMCF10CA1a M2 المشتقة (M4) ، الذي أنشئ من M2 xenografts وأشكال سرطان الدرجة العالية ، مع القدرة على metastasize إلى 9 في الرئة. هذه السلسلة MCF10A توفر إمكانية لدراسة استجابات الخلايا مع درجات مختلفة من الأورام الخبيثة التي ليست منحازة بواسطة خلفية وراثية مختلفة. لتحليل TGF - β بفعل الغزو ، ولدت لنا MCF10A كروي مثلي النمط embe الثقافات الخليةdded في مصفوفة 3D الكولاجين في المختبر (الشكل 1). مثل هذه النماذج البشرية تشبه الأورام في الجسم الحي من خلال إنشاء التدرج من الاوكسجين والمواد الغذائية ، مما أدى إلى خلايا نشطة والغازية على الخلايا خارج وهادئة أو حتى نخرية في الداخل من 10 كروي. كما تم عرض كروي المقايسات تستند إلى أفضل تلخيص مقاومة المخدرات من الثقافات أحادي الطبقة 11. يسمح هذا النموذج 3D MCF10 نظام لنا لتحقيق تأثير TGF - β يدل على خصائص الغازية من خلايا الثدي في مراحل مختلفة من السرطان.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

Spheroid Assay to Measure TGF-&#946;-induced Invasion

يوصف لفحص لقياس كميا تحويل عامل النمو (TGF) - β بفعل الغزو في المواد الهلامية الكولاجين 3 الأبعاد. هذا الاختبار يستفيد من سلسلة MCF10A من خطوط الخلايا التي تمثل مراحل مختلفة من تطور مرض السرطان الثدي. ويمكن اعتماد هذه الطريقة ليتم استخدامها مع خطوط الخلايا الأخرى ، ويمكن استخدامها لتفعيل التحقيق المحتملين الآخرين أو مثبطات الغزو.

كروي الفحص لقياس TGF - β بفعل الغزو

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

spheroid-assay-to-measure-tgf-induced-invasion

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21.8K Views.  Leiden University Medical Centre. يوصف لفحص لقياس كميا تحويل عامل النمو (TGF) - β بفعل الغزو في المواد الهلامية الكولاجين 3 الأبعاد. هذا الاختبار يستفيد من سلسلة MCF10A من خطوط الخلايا التي تمثل مراحل مختلفة من تطور مرض السرطان الثدي. ويمكن اعتماد هذه الطريقة ليتم استخدامها مع خطوط الخلايا الأخرى ، ويم...

Video: Spheroid Assay to Measure TGF-β-induced Invasion

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

في الوقت الحقيقي المعاوقة الكهربائية تقنية تستند إلى قياس غزو أحادي الطبقة الخلية البطانية من قبل خلايا السرطان

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

النمذجة والتصوير 3 الأبعاد غزو الخلية الجماعية

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

طريقة لقياس النغمة من العضلات المحورية والدانية

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

الدقة في طب الأسنان، طريقة جديدة لقياس الصدق والدقة

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

والتعديل<em&gt; في المختبر</em&gt; الغزو الفحص لتحديد الدور المحتمل للالهرمونات، السيتوكينات و / أو عوامل النمو في التوسط السرطان غزو الخلايا

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

تقييم سرطان المبيض كروي مرفق وغزو الخلايا الظهارية في الوقت الحقيقي

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

القياس عن بعد داخل الرحم لقياس ماوس مقلص الضغط<em&gt; في فيفو</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

ثلاثي الأبعاد (3D) ورم الجسم الشبه الكروي الغزو الفحص

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

فرخ القلب الغزو الفحص لاختبار الغازية من خلايا سرطان وآخر للمركبات يحتمل مكافحة الغازية

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

خلية السرطان كروي الفحص لتقييم الغزو في إعداد 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

كروي الفحص لقياس TGF - β بفعل الغزو

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