Name: antibody transfection into neurons as a tool to study disease pathogenesis
Uploaded: 2012-09-26T13:00:00
Description: Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

ويدعم هذا العمل من قبل مكتب البحوث والتنمية، الخدمات الطبية البحوث وزارة شؤون المحاربين القدامى. وقد تم تمويل هذه الدراسة من قبل على جائزة الاستحقاق استعراض VA (لMCL) وجامعة ولاية تينيسي العلوم الصحية مركز التصلب المتعدد صندوق البحث.

1. وصفها الأجسام المضادة (الشكل 1) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل 0.1 M 3 NaHCO العازلة: <ul type="circle" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 8،4 ز NaHCO 3 </li><li style=";text-align:right;direction:rtl"> 29،2 ز كلوريد الصوديوم </li><li style=";text-align:right;direction:rtl"> 1 لتر H 2<...

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من أجل اختبار الفرضيات حول جزيئات معينة والتعبير عنها، وتقاس عادة الجينات وناقلات أخرى في الخلايا. الجانب الفريد من هذا الإجراء هو القدرة على الأجسام المضادة في بالنقل بسهولة الخلايا المستهدفة. أجرينا هذه الطريقة في خمس تجارب منفصلة في كل التكرارات. وكانت النتائج م?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> كتالوج رقم </td></tr><tr><td> مجهر الفلورسنت AxioObserver A1 </td><td> زايس </td><td></td></tr><tr><td> Axiovision الإصدار 4.2 </td><td> زايس </td><td></td></tr><tr><td> مكافحة rhnRNPA1 </td><td> Abcam </td><td> Ab4791 </td></tr><tr><td> FITC المسمى مكافحة ريج </td><td> OZ العلوم البيولوجية </td><td> AI20100 </td></tr><tr><td> Atto550 NHS استر </td><td> سيجما </td><td> 92835 </td></tr><tr><td> AB-DeliverIN </td><td> OZ العلوم البيولوجية </td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10٪ FBS المضادات الحيوية +1٪ </td><td> Gibco </td><td> 11320-033 </td></tr><tr><td> بولي يسين D-8-الشرائح الثقافة جيدا </td><td> BD </td><td> 354632 </td></tr><tr><td> متعددة الأغراض الدوار </td><td> العلمية والصناعاتوفاق شركة </td><td> نموذج 151 </td></tr><tr><td> دابي التركيب وسائل الإعلام </td><td> ميليبور </td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion </td><td> 9626 </td></tr><tr><td> مديال </td><td> الحرارية فيشر العلمية </td><td> 66380 </td></tr><tr><td> Amicon الترا 0.5- </td><td> ميليبور </td><td> UFC501096 </td></tr><tr><td> بارافورمالدهيد (الحل 4٪) </td><td> علوم المجهر الإلكتروني </td><td></td></tr><tr><td> نانو قطرة </td><td> الحرارية فيشر العلمية </td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

ووصف النهج السريع للتحقيق في التفاعلات والآثار على الآليات الجزيئية ذات الصلة وجود الأجسام المضادة في بيئة داخل الخلايا. الأسلوب ينطوي ترنسفكأيشن من الأجسام المضادة في الخلايا الحية باستخدام تشكيل التساهمية غير معقدة تقوم على صياغة المادة الدهنية. هذه التقنية قابلة للتكيف مع خطوط الخلايا والخلايا الأولية خلد.

ترنسفكأيشن الأجسام المضادة إلى الخلايا العصبية كأداة للدراسة إمراض مرض

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

القبض على الليزر Microdissection ذبابة الفاكهة الخلايا العصبية المحيطية

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

خفض التحميل : أداة مفيدة لفحص مدى تقاطع الفجوة بين الخلايا العصبية توصيلات الراسم الشبكية

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

الكالسيوم فوسفات ترنسفكأيشن من الخلايا العصبية الحصين الابتدائية

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

محلل خلية مقرها الممانعة الحقيقي في الوقت كأداة لتحديد المسارات الجزيئية تشارك في العصبية والحمايه العصبيه في خط الخلية العصبية

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

النظام الشمي كنموذج لدراسة أنماط نمو محور عصبي ومورفولوجيا<em&gt; في فيفو</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

الحقن في العضل وإلى جانب لوحات السيارات نهاية: نهج المناظير لمكوك الراسمات مباشرة إلى الخلايا العصبية للسيارات

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

شريط الفحص لدراسة آخر جذاب أو مثيرة للاشمئزاز من الركيزة البروتين عن طريق فصل الخلايا العصبية الحصين

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

أورجانوتيبيك شريحة هيبوكامبال الثقافات كنموذج للدراسة نيوروبروتيكتيون واختزاع الخلايا السرطانية

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

إدارة خارجية من المجاميع المرتبطة ميكروسوميس ألفا-سينوكلين إلى الخلايا العصبية الأولية كنموذج خلية قوية لتشكيل ييفات

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

تحليل يستند إلى الخلية لدراسة التخليص تاو جسم بوساطة من Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...