وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح R01-GM-098977-R21 وAI-098122 لبنك الكويت المركزي وKEG. وأيد RSS في جزء من زمالة مؤسسة لوسي وجزئيا على زمالة برنامج الابتكار ثاير ثاير من كلية الهندسة.

<ol>
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يعمل ليونارد مويس قبل ويحمل خيارات الأسهم في EpiVax، وهي شركة مملوكة للقطاع الخاص شركة للتكنولوجيا الحيوية تقع في بروفيدانس، رود ايلاند. العمل الواردة في هذا التقرير البحثي خالية من أي تحيز التي قد تترافق مع الأهداف التجارية للشركة.

وقد أنشأت Biotherapeutics نفسها باعتبارها حجر الزاوية في الطب الحديث، وهو ما يمثل أربعة من الخمسة الاوائل الأدوية مبيعا في عام 2012 32. وقد أظهرت قطاع الصيدلانيات البيولوجية النمو المطرد لعدة سنوات 6، والتطوير المستمر للعوامل الرواية فضلا عن ظهور البدائل الحيوية و...

An erratum was issued for: <a href="https://www.jove.com/video/51308/a-high-throughput-mhc-ii-binding-assay-for-quantitative-analysis">A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes</a>.&#160; There were typos in the Protocol section and Table 2.
Step 3.1.1 in the Protocol was updated from:
Add 80 &#181;l of 1 mM Pefa Bloc and 60 mg of octyl-&#946;-D-glucopyranaside to 3,920 &#181;l of Citrate Phosphate Buffer. (See supplemental spreadsheet calculator for alternative scaling).
to:
Add 80 &#181;l of 1 mM Pefa Bloc and 60 &#181;g of octyl-&#946;-D-glucopyranaside to 3,920 &#181;l of Citrate Phosphate Buffer. (See supplemental spreadsheet calculator for alternative scaling).
Step 5.2&#160;in the Protocol was updated from:
Further dilute the Control Peptide from step 5.1 (20 mM) 1:100 into the MHC II Master Mix from step 3.2.
to:
Further dilute the Control Peptide from step 5.1 (20 &#181;M) 1:100 into the MHC II Master Mix from step 3.2.
Table 2 was updated from:
<table border="0" cellpadding="0" cellspacing="0" width="376">
	<tbody>
		<tr>
			<td>MHC II Allele DRB1*:</td>
			<td>1501</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mg/ml)</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mM)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Vol. MHC II Stock to Add (ml)</td>
			<td>14.65</td>
		</tr>
		<tr>
			<td>Vol. Reaction Buffer to Add (ml):</td>
			<td>2885.35</td>
		</tr>
		<tr>
			<td>MHC II Master Mix Concentration (nM)</td>
			<td>101</td>
		</tr>
	</tbody>
</table>
to:
<table border="0" cellpadding="0" cellspacing="0" width="376">
	<tbody>
		<tr>
			<td>MHC II Allele DRB1*:</td>
			<td>1501</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mg/ml)</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (&#956;M)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Vol. MHC II Stock to Add (&#956;l)</td>
			<td>14.65</td>
		</tr>
		<tr>
			<td>Vol. Reaction Buffer to Add (&#956;l):</td>
			<td>2885.35</td>
		</tr>
		<tr>
			<td>MHC II Master Mix Concentration (nM)</td>
			<td>101</td>
		</tr>
	</tbody>
</table>

An erratum was issued for: <a href="https://www.jove.com/video/51308/a-high-throughput-mhc-ii-binding-assay-for-quantitative-analysis">A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes</a>.&#160; There were typos in the Protocol section and Table 2.

Erratum: A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

البروتينات هي أسرع فئة متزايدة من العوامل العلاجية 6، وركزت التوسع السريع في خطوط الأنابيب biotherapeutic باهتمام متزايد على التحديات المرتبطة بالتنمية واستخدام العقاقير البروتين. ينبع اعتبار واحد فريد من نوعه من حقيقة أنه، في نظام المناعة الصحي والعمل، يتم أخذ عينات جميع البروتينات خارج الخلية بواسطة مستضد تقديم الخلايا (ناقلات الجنود المدرعة). مرة واحدة المنضوية بواسطة ناقلات الجنود المدرعة، والمشقوق بروتين الببتيد إلى شظايا صغيرة، ويتم تحميل شرائح المناعية المفترضة في أخدود من الدرجة الثانية البروتينات التوافق النسيجي الرئيسي المعقدة (MHC II). ثم يتم عرض المجمعات الببتيد-MHC II على سطح APC، والببتيدات مناعة حقيقية، ووصف خميس الحواتم الخلايا، وشكل الثلاثي MHC II-الببتيد-T المجمعات مستقبلات الخلايا CD4 T مع المشابهة مستقبلات سطح الخلية 7. هذا الحدث الاعتراف الجزيئية الحرجة يبدأ سلسلة معقدة إشارات، مما يؤدي إلى تنشيط الخلايا T، والإفراج عن خلوىق، CD4 T-B بوساطة خلية خلية النضج، وفي نهاية المطاف إنتاج الأجسام المضادة المنتشرة التي تربط مفتش لمسح والخارجية البروتين المتسبب في المشكلة. وبالتالي، قد يكون deimmunized البروتينات المناعية عن طريق تحديد التأسيسية الحواتم الخلايا التائية وتحور بقايا الرئيسية المسؤولة عن تشكيل MHC II معقدة. مشيرا إلى أنها تتحمل، مع ذلك، أن خميس الحواتم الخلية يمكن أن تكون عديدة وعلى نطاق واسع في جميع أنحاء توزيع البروتينات المناعية، والغالبية العظمى من الطفرات حذف حاتمة من المحتمل أن يسبب خسائر غير مقصودة من وظيفة البروتين أو الاستقرار. وبالتالي، يمكن deimmunized الهندسة biotherapies أن يكون هدفا معقدة وصعبة من الناحية الفنية، ولكن توجد العديد من الأمثلة الناجحة T حاتمة خلية المشاريع الحذف 3،5،8-12. على عكس القائمة على تطعيم &quot;أنسنة&quot;، الذي يقتصر إلى حد كبير إلى العلاجات الضد، حاتمة الحذف يمكن تطبيقها على أي هدف أساسا من البروتين بغض النظر عن تسلسل، وهيكل، وظيفة، أو توافر homologoلنا السقالات الإنسان. الخطوة الأولى لتنفيذ هذا النهج هو تحديد الحواتم الببتيد الرئيسية جزءا لا يتجزأ من داخل تسلسل البروتين الهدف. يمكن عالية الإنتاجية فحوصات البيوكيميائية باستخدام الببتيدات الاصطناعية والمؤتلف الإنسان جزيئات MHC II تقديم رؤى الأولية السريعة في تحديد حاتمة والتخفيف 1،3-5. ويمكن لهذه المقايسات نوع ELISA يكون مكملا قويا لغيرها من تصميم وتطوير الأدوات البروتين / قاح. على سبيل المثال، واحدة المنهج التجريبي راسخة لرسم خرائط حاتمة يعتمد على الوقت، والعمل، والموارد فيفو السابقين المقايسات تكاثر الخلايا المركزة 15. لفترة وجيزة، وينقسم التسلسل الرئيسي للبروتين الهدف الأول في لوحة من الببتيدات متداخلة، وغالبا 15 السائدة مع 12 بقايا تتداخل بين الببتيدات المجاورة. يتم تصنيعه كيميائيا لوحة الببتيد ويتم اختبار المناعية لكل الببتيد في واحدة من عدة المناعية المختلفة التي توظف bloo الطرفيةالخلايا وحيدة النواة د (PBMC) المعزولة من المانحين الإنسان 13،14. لتوفير قدر أكبر من الثقة في النتائج، يتم اختبار الببتيدات عادة في تكرار مع PBMC من 50 أو أكثر الجهات المانحة المختلفة. في الحالات التي يكون فيها deimmunization هو الهدف النهائي، وتتفاقم الأعمال الأخرى بسبب الحاجة لإنتاج لوحات إضافية من الببتيدات تحور واختبار الألواح الببتيد الجديدة في المقايسات PBMC قبل إدخال أي طفرات في بروتين deimmunizing طول الكامل للتحليل وظيفي لاحقة 10. في حين لا تزال هذه المقايسات الخلوية المعيار الذهبي لتقييم إمكانات المناعية في المرضى من البشر، يمكن تحسين كفاءة هذا النهج شاملة من قبل prefiltering الحواتم المناعية المفترضة باستخدام الإنتاجية السريعة وعالية MHC II-الببتيد مقايسة ملزمة. وبالمثل، والكيمياء الحيوية المقايسات ملزمة الببتيد-MHC II يمكن دمجها مع التنبؤية في أساليب سيليكون لتسريع جذري في عملية تحديد الهوية حاتمة.توجد مجموعة متنوعة من الأدوات الحسابية لحاتمة تي خلية التنبؤ، وتشمل الأمثلة ProPred 16، MHCPred 17، SVRMHC 18، ARB 19، SMM محاذاة 20، 21 NetMHCIIpan فضلا عن أدوات الملكية مثل EpiMatrix بواسطة EpiVax 22. وبالمثل، تم مؤخرا جنبا إلى جنب مع تنبؤ حاتمة المعلوماتية الحيوية الأخرى، وأدوات النمذجة الجزيئية لانتاج خوارزميات deimmunization البروتين متكاملة تهدف للحد من المخاطر التي قد الطفرات deimmunizing تعطيل بنية البروتين وظيفة 23-26. في حين أثبتت عدة تنبئ حاتمة أن تكون دقيقة إلى حد معقول 27،28، النتائج الحسابية تتطلب دائما التحقق التجريبي. السريع، وإنتاجية عالية، وفعالة من حيث التكلفة الطرق التجريبية هي الأنسب كعامل تصفية أولية للسيليكون في التنبؤات حاتمة. في سياق مماثل، يمكن أن تنبئ حاتمة مستضد لاختيار قيادة vaccinolo العكسيغراي 29،30. على سبيل المثال، قد أسفرت عن التقدم في المعلوماتية الحيوية شاشات الجينوم التي تحدد بسرعة اللقاحات المرشحة في شكل البروتينات كليا أو الحواتم الببتيد المستخرجة من proteomes الممرض. بينما هذه التكنولوجيا تمكن هو إعادة تشكيل اكتشاف وتطوير لقاحات واقية، فإنه يدخل تحديا جديدا في شكل قوائم كبيرة intractably المرشحين لقاح مناعة. يمكن المقايسات ملزمة عالية الإنتاجية الببتيد-MHC II توجيه اختيار حاتمة عن طريق قياس الببتيد تقارب ملزمة ملزمة والمجون بين الأليلات متعددة MHC II. كما هو الحال مع البروتين deimmunization، فإن المطلوب من هذه الطرق التجريبية في نهاية المطاف للتحقق من صحة التنبؤات الحسابية يؤدي لقاح واعد. هنا، يوصف مقايسة ملزمة الببتيد-MHC II تحجيمها إلى تنسيق 384 جيدا. البروتوكول هو متوازية إلى حد كبير ويقلل من التكاليف بنسبة 75٪ كاشف مقارنة موضح سابقا 96 لوحة جيدا صيغ 1،3-5. باستخدام LIQUI واحدد التعامل مع الروبوت، وهذا الأسلوب يسمح أحد الباحثين لتحليل ما يقرب من تسعين الببتيدات بسهولة اختبار في ثلاث نسخ على مجموعة من ثمانية تجمعات وأربعة أنواع أليل MHC الثاني في أقل من 48 ساعة. توضح هذه المقالة الإعداد من لوحة واحدة 384 جيدا لتحليل ELISA سبعة الببتيدات التجريبية ضد أحد MHC II الأليل، ولكن يتم توفير الآلات الحاسبة ورقة انتشرت كمادة تكميلية بحيث يسهل على مقياس التجربة إلى أي عدد من الببتيدات المطلوب و / أو MHC الثاني الجزيئات.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:587px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:357px;">Sigma</td>
			<td style="width:217px;">221732</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Citric Acid</td>
			<td>Sigma</td>
			<td>C1901</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dibasic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trizma HCl</td>
			<td>OmniPur</td>
			<td>9310</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Tween-20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">100% DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Octyl-&#946;-D-Glucopyranaside</td>
			<td>Fischer</td>
			<td>29836-26-8</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Pefa Bloc SCF</td>
			<td>Roche</td>
			<td>1158591600</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dulbecco&#8217;s 10X Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14200-166</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Assay Buffer</td>
			<td>Perkin Elmer</td>
			<td>4002-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Enhancement Buffer</td>
			<td>Perkin Elmer</td>
			<td>4001-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Europium Labelled Streptavidin</td>
			<td>Perkin Elmer</td>
			<td>1244-360</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">L243 anti-HLA-DR antibody</td>
			<td>Biolegend</td>
			<td>307602</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Biotinylated tracer peptides</td>
			<td>21st Century Biochemicals</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Test peptides (1-4mg, 85% purity)</td>
			<td>Genscript</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Purified HLA-DRB1 monomers (non-biotinylated)</td>
			<td>Benaroya Research Institute*</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">384-well white EIA/RIA plate</td>
			<td>Thermo</td>
			<td>460372</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Polypropylene 384-well plate</td>
			<td>Costar</td>
			<td>3656</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Film, AxySeal, 80 &#181;m, ELISA Applicator&#160;</td>
			<td>Axygen Asicentific</td>
			<td>PCR-SP</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">MilliQ Water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Epimotion Liquid Handler (or similar)</td>
			<td>Eppendorf</td>
			<td>5075</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Select TS Plate Washer (or similar)</td>
			<td>BioTek</td>
			<td>405</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">SpectraMax Gemini Microplate Reader (or similar)</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4" height="98" style="height:99px;width:1161px;">*Recombinant human MHC II molecules can be obtained from the Benaroya Tetramer Core Laboratory. See: https://www.benaroyaresearch.org/our-research/core-resources/tetramer-core-laboratory</td>
		</tr>
	</tbody>
</table>

a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes

يمكن فحوصات البيوكيميائية مع المؤتلف الإنسان جزيئات MHC II تقديم سريع، رؤى الكمي في تحديد حاتمة المناعية، الحذف، أو تصميم 1،2. هنا، يتم تحجيم مقايسة ملزمة الببتيد-MHC II إلى تنسيق 384 جيدا. بروتوكول تقليص يقلل التكاليف بنسبة 75٪ كاشف والإنتاجية أعلى من البروتوكولات التي سبق وصفها جيدا 1،3-5 96. على وجه التحديد، والتصميم التجريبي يسمح تحليل دقيق وقابلة للتكرار تصل إلى 15 الببتيدات ضد واحد أليل MHC II في 384 جيدا لوحة ELISA. باستخدام السائل واحد التعامل مع الروبوت، وهذا الأسلوب يسمح أحد الباحثين لتحليل ما يقرب من تسعين الببتيدات اختبار في ثلاث نسخ على مجموعة من ثمانية تجمعات وأربعة أنواع أليل MHC الثاني في أقل من 48 ساعة. غيرهم من العاملين في مجالات البروتين deimmunization أو تصميم وتطوير لقاح قد تجد البروتوكول أن تكون مفيدة في تسهيل عملهم. على وجه الخصوص، وتعليمات خطوة بخطوة والشكل البصريمن إن الرب يجب السماح للمستخدمين الآخرين بسرعة وسهولة لإنشاء هذه المنهجية في مختبرات خاصة بهم.

تم تحليل تسلسل الببتيد ناضجة من الأمعائية المذرقية P99 بيتا اكتاماز (جيش تحرير بلوشستان) (بنك الجينات رقم # X07274.1) مع ProPred 16 لدنة الببتيد المفترضة لMHC II أليل DRB1 * 1501 (جدول 4). حددت ProPred 117 الببتيدات nonamer مع إلزامية P1 مرساة بقايا (أي M، L، I، V، F، Y، W أو في ا...

Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

يمكن فحوصات البيوكيميائية مع المؤتلف الإنسان جزيئات MHC II تقديم سريع، رؤى الكمي في تحديد حاتمة المناعية، الحذف، أو التصميم. هنا، يوصف مقايسة ملزمة الببتيد-MHC II تحجيم لوحات 384 جيدا. هذا الشكل فعالة من حيث التكلفة يجب أن تكون مفيدة في مجالات deimmunization البروتين وتصميم لقاح والتنمية.

A تجليد الفحص إنتاجية عالية MHC II لتحليل الكمي لالببتيد الحواتم

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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Research

JoVE Journal

Biology

14.8K Views.  Dartmouth College.  يمكن فحوصات البيوكيميائية مع المؤتلف الإنسان جزيئات MHC II تقديم سريع، رؤى الكمي في تحديد حاتمة المناعية، الحذف، أو التصميم. هنا، يوصف مقايسة ملزمة الببتيد-MHC II تحجيم لوحات 384 جيدا. هذا الشكل فعالة من حيث التكلفة يجب أن تكون مفيدة في مجالات deimm...

Video: A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

المستضد CD4 + تصور معين باستخدام خلايا T MHC الدرجة الثانية Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

والخلايا العصبية ونجمية المشارك الثقافة الفحص لتحليل محتوى عال من العصبية

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

الإنتاجية العالية للمواد الفحص تسكر Lignocellulosic

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

تحليل كمي لياقة سير العمل

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

الإنتاجية العالية فحص وظيفية باستخدام محلية الصنع ثنائي توهج luciferase الفحص

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

تدفق تحليل Cytometric من التكملة الإسفار ثنائي الجزيء: عالية الإنتاجية الأسلوب الكمي لدراسة التفاعل البروتين البروتين

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

الإنتاجية العالية للكشف عن المغيرون الكيميائية من الجينات المشاركة ما بعد transcriptionally الخاضعة للرقابة

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

تحليل qPCRTag - إنتاجية عالية، في الوقت الحقيقي PCR الفحص لSc2.0 التنميط الجيني

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

عالية الإنتاجية<em&gt; دانيو Rerio</em&gt; طاقة الإنفاق الفحص

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...