이 작품은 연구 개발의 사무실, 의료 연구 서비스, 보훈학과에서 지원됩니다. 이 연구는 VA 공훈 검토 상 (MCL에)와 테네시 건강 과학 센터 다발성 경화증 연구 기금의 대학 재정 지원되었다.

1. 항체 라벨 (그림 1) <ol><li> 0.1 M NaHCO 3 버퍼를 만들기 : <ul type="circle"><li> 8.4 g NaHCO 3 </li><li> 29.2 g NaCl </li><li> 1리터 H 2 O </li></ul></li><li> 이 솔루션 (10.6 g 오세영 2 CO 3, 29.2 g NaCl, 1리터 H 2 0)과 8.4의 산도에 버퍼를 가져와. </li><li> 70 μl 방지 hnRNPA1 500 μl NaHCO 3 버퍼 35 μl Atto550 NHS를 추가하고 실온에서 1 시간에 회전 할 수 있습니다. 1 시간 긴 회전 후, dialyzer로 혼합물을 주입하고 하루 아침에 PBS 2 리터에 dialyze. </li><li> 다음 날, 스핀 컬럼 (Amicon 울트라 0.5)를 사용하여 안티 - hnRNPA1 dialyzed Atto550 NHS을 집중. </li><li> Atto550 NHS는 반 hnRNPA1 항체가 집중되어 표시 한 후, 나노 드롭 샘플 농도를 결정합니다. </li></ol> 2. 항체 Transfection (그림 2) <ol><li> Dulbecc의 씨 10 5 세포 /도 500에 μl이전 transfection로 8도 슬라이드 24 시간의 오의 수정 된 이글 매체 (DMEM/F12 10% FBS 1% 항생제). 셀 confluency는 최소 70 %의합니다. </li><li> 배란 후 24 시간 시딩 후 Eppendorf 튜브에 AB-DeliverIN 시약 2 μl를 추가합니다. </li><li> 다음, 같은 Eppendorf 튜브에 방지 hnRNPA1 항체 분류 Atto550 NHS 2 μg을 (0.5 μg / μl)을 추가하고 RT로 10-15 분 알을 품다. </li><li> 부화하는 동안 미디어를 기음과 세포에 신선한 DMEM 매체 394 μl를 추가합니다. </li></ol> 참고 : 컨트롤 역할을 손길이 닿지 않은 셀 중 하나 챔버에 500 μl DMEM을 추가합니다. <ol start="5"><li> 부화 후, 항체 혼합물에 DMEM 100 μl를 추가 아래로 pipetting과에 의해 혼합, 500 μl의 총 볼륨 DMEM에있는 셀에 추가 할 수 있습니다. </li><li> 항체 전달의 48 시간 후 미디어를 변경합니다. </li></ol> 참고 : 프로토콜은 FITC 긍정적 인 제어로 rIgG 표시를 반복했다. 3. 효율성을 결정하기 위해 라이브 및 고정 이미징 <ol><li> 이미지 셀 이미지 transfected Atto550 NHS이라는 항체를 할 수있는 형광 현미경에 Cy3 필터를 사용하여 48 시간에 살고 있습니다. </li><li> 라이브 영상이 완료되면, 미디어 및 수정 전지를 대기음. </li><li> aspirating 미디어 후, 실온에서 15 분에 0.4 %의 Paraformaldehyde과 세포를 취급합니다. 세포에게 4 X 5 분마다 PBS로 씻는다. 그런 다음 DAPI 시약 및 수정 coverslip을 포함 장착 미디어와 세포를 탑재합니다. Axiovision 소프트웨어와 고정 세포의 transfection 효율을 측정합니다. Axiovision 소프트웨어가 손에 없으면, 단순히 3.7에서 계산에 손 플러그 결과하여 이미지에서 이벤트를 계산합니다. 또한, ImageJ 소프트웨어는이 계산에 사용할 수 있습니다. </li><li> 첫째, 20x 확대에 원하는 이미지에 세포의 총 수를 계산하려면 &quot;이벤트&quot;측정을 사용합니다. </li><li> 다음 아니었다 세포 수를 계산하려면 &quot;이벤트&quot;측정을 사용하여성공적으로 동일한 20x 배율 이미지의 항체와 transfected. </li><li> 엑셀 스타일의 형식으로 측정 된 &quot;이벤트&quot;에 액세스하려면 &quot;속성&quot;을 클릭 &quot;측정&quot;탭을 클릭하고 확인을 클릭합니다 &quot;테이블.&quot; </li><li> 마지막으로, 두 이전에 측정 이벤트를 비교하여 transfection 효율을 결정하기 위해 다음과 같은 계산을 사용합니다 ((총 세포 - Untransfected 셀) / 총 전지) × 100 = Transfection 효율. </li></ol> 4. 대표 결과 untransfected의 라이브 이미지 (그림 3A)이 transfected (그림 3B) 뉴런에 비해 것은 항체의 라벨이 (각 그림에서 빨간색 또는 녹색) 명확하게 세포 (그림 3B, C, D)에서 관찰되는 알 수있다. 흡인에 따라 사이 항체 또는 세포의 표면에 점착성의를 제거하는 미디어와 세탁기, 표시된 항체는 여전히 뉴런 (그림 3C, D)에 존재한다.<html> transfection 효율을 계산하기 위해 성공적으로 transfected되지 않은 세포에 비해 총 셀을 계산 Axi​​ovision 소프트웨어를 사용합니다. 이 값은 전체 세포의 비율 (그림 4)로 transfection 효율을 확보 할 수있는 transfection 효율 공식을 체결했다. <img alt="그림 1" src="/files/ftp_upload/4154/4154fig1.jpg" /> 그림 1. 아토 550 NHS 에스테르와 라벨 항체에 사용되는 절차의 흐름 다이어그램 라벨 항체을 집중하는 데 필요한 단계 다음은. <a href="http://www.jove.com/files/ftp_upload/4154/4154fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img alt="그림 2" src="/files/ftp_upload/4154/4154fig2.jpg" /> 그림 2. 문화의 라이브 셀에 표시된 항체를 transfect하는 데 사용되는 절차의 흐름 다이어그램.arge.jpg &quot;대상 =&quot;_blank &quot;&gt; 큰 그림을 보려면 여기를 클릭하십시오. <img alt="그림 3" src="/files/ftp_upload/4154/4154fig3.jpg" /> 그림 3. transfected 뉴런 48 시간 다음 transfection의 이미지를 살고 있습니다. <img alt="그림 4" src="/files/ftp_upload/4154/4154fig4.jpg" /> 그림 4. transfection 효율을 결정하기 위해 계산 이벤트와 이미지를 수정하고 헹구어.

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관심 없음 충돌이 선언 없습니다.

특정 분자와 표현에 대한 가설을 테스트하기 위해, 유전자 및 기타 벡터는 일반적으로 세포로 transfected 수 있습니다. 이 절차의 독특한 측면은 쉽게 대상 세포에 항체를 transfect 할 수있는 능력입니다. 우리는 5 개의 실험을 중복의 각이 방법을 수행했습니다. Transfection 효율 결과는 모든 실험들과 비슷했다. 중요한 방법은 항체의 구조 나 기능을 변경하지 않고 셀에 항체의 항목을 수 있습니다. 또?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 형광 현미경 AxioObserver A1 </td><td> Zeiss </td><td></td></tr><tr><td> Axiovision 버전 4.2 </td><td> Zeiss </td><td></td></tr><tr><td> 안티 rhnRNPA1 </td><td> Abcam </td><td> Ab4791 </td></tr><tr><td> FITC 안티 rIgG 표시 </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> Atto550 NHS 에스테르 </td><td> 시그마 </td><td> 92,835 </td></tr><tr><td> AB-DeliverIN </td><td> OZ Biosciences </td><td> AI20100 </td></tr><tr><td> DMEM/F12 10%는 FBS 1%의 항생제 </td><td> Gibco </td><td> 11320-033 </td></tr><tr><td> 폴리-D-라이신 8도 문화 슬라이드 </td><td> BD </td><td> 354632 </td></tr><tr><td> 다중 목적 회전하는 것 </td><td> 과학 Industri에스 주식회사 </td><td> 모델 151 </td></tr><tr><td> DAPI 장착 미디어 </td><td> Millipore </td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion </td><td> 9626 </td></tr><tr><td> Dialyzer </td><td> 온도 피셔 과학 </td><td> 66,380 </td></tr><tr><td> Amicon 울트라 - 0.5 </td><td> Millipore </td><td> UFC501096 </td></tr><tr><td> Paraformaldehyde (4 % 용액) </td><td> 전자 현미경 과학 </td><td></td></tr><tr><td> 나노 드롭 </td><td> 온도 피셔 과학 </td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

항체가 생활 시스템에서 일어나는 다양한 이벤트에 소설 통찰력을 얻을 수있는 기능을 제공합니다. 대상 단백질에 매우 구체적인 항체를 생산하는 능력은 해결 될 매우 정확한 생물학적 질문에 사용할 수 있습니다. 중요한 항체는 전신 홍반 루푸스 (SLE), 류마티스 관절염 (RA), paraneoplastic syndromes, 다발성 경화증 (MS)와 인간 T-lymphotropic 바이러스 타입 1 (HTLV-1)를 포함한 인간의 질병의 숫자의 pathogenesis에 연루되어 myelopathy / 열대 경련 paraparesis (햄 / TSP) 1-9 연결된 상태입니다. 항체는 질병이 진행중인 조사의 영역이며, 데이터 항체와 염증의 다양한 세포 내 분자 결과 사이의 상호 작용은 세포 메시징 및 apoptosis 10 변경하는 것이 좋습니다 원인이 방법. 그것은 MS와 햄 / TSP 환자는 세포 내 RNA 결합 단백질 이기종 ribonuclear 단백질에 autoantibodies을 생산하는 것으로 나타되었습니다1월 5일에서 7일까지 (hnRNP A1) 3, 9, 11. 최근 데이터 간 neuronal 및 표면 항원 모두 항체가 병원성 3, 5-9 11아르을 나타냅니다. 따라서, 세포 내 항체의 연구를 위해 할 수있는 절차 : 단백질 상호 작용은 질병 pathogenesis에 큰 통찰력을 부여합니다. 유전자는 일반적으로 기본 세포와 문화에 세포 라인에 transfected 있으며, 세포에 항체의 그러나 transfection이 항체 구조 또는 낮은 transfection 효율을 12 개조에 의해 방해했습니다. transfection의 다른 방법은 양이온 liposomes (DOTAP / 마약으로 구성)과 polyethylenimines (PEI)을 기반으로 항체 transfection을 포함, 컨트롤 12에 비해 항체 transfection의 열 배나 감소로 이어진 둘 다. 우리의 연구에서 수행 한 방법은 양이온 지질로 인한 방법과 유사하고를 통해 항체와 비 공유 결합 복합체를 형성하기 위해 지질 기반 메커니즘을 사용 정전기 및 hydrophobic 상호 작용 13. 우리는 비 공유 결합 정전기와 소수성 상호 작용을 통해 항체를 캡쳐하고 세포 내부를 제공 할 수있는 지질 제제입니다 AB-DeliverIN 시약을 이용. 따라서 화학 및 유전 커플 링은 생활 세포에 기능 항체의 전달에 필요하지 않습니다. 단백질 상호 작용 9 :이 방법은 우리가 다양한 항체는 단백질 현지화 실험뿐만 아니라, 세포 내 항체의 분자 결과의 분석을 추적하고 수행 할 수있게되었습니다. 이 프로토콜에서는, 우리는 빠르게, reproducibly과 transfection 효율의 높은 수준의 뉴런에 항체를 transfect하는 방법을 보여줍니다. 예를 들어, 우리는 반 hnRNP A1 및 안티 IgG 항체를 사용합니다. transfection 효율을 쉽게 정량화를 들어 우리는 아토-550-NHS와 FITC-라벨 IgG으로 표시 방지 hnRNP A1 항체를 사용했습니다. Atto550 NHS는 고분자 흡수 및 양자 순으로 새 레이블연령. 여기 소스와 Atto550에 대한 형광 필터는 Cy3 (예 556 엠. 578)과 비슷합니다. 또한, Atto550 높은 photostability 있습니다. FITC-라벨 IgG는이 방법에 의존 염색 다목적 및 아니라는 것을 보여주기 위해 제어로 사용되었습니다. 이 방법과 생성 된 데이터는 대상 항원을 세포하기 위해 항체 인간 질병의 pathogenesis에서 재생 할 수있는 역할의 이해에 도움이 될 것입니다.

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

상호 작용과 세포 내 환경에서 항체의 존재에 관한 분자 메커니즘에 미치는 영향을 조사하기 위해 신속하게 접근 방식이 설명되어 있습니다. 방법은 지질 공식에 따라 비 공유 결합 복잡한 형성을 사용하여 라이브 세포에 항체의 transfection을 포함한다. 기술은 불후의 세포 라인과 기본 셀에 적용 할 수 있습니다.

질병 Pathogenesis를 공부하는 도구로 뉴런에 항체 Transfection

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

antibody-transfection-into-neurons-as-tool-to-study-disease

Research

JoVE Journal

Neuroscience

10.7K 조회수.  Veterans Administration Medical Center, Memphis, TN. 상호 작용과 세포 내 환경에서 항체의 존재에 관한 분자 메커니즘에 미치는 영향을 조사하기 위해 신속하게 접근 방식이 설명되어 있습니다. 방법은 지질 공식에 따라 비 공유 결합 복잡한 형성을 사용하여 라이브 세포에 항체의 transfection을 포함한다. 기술은 불후의 세포 라인과 기본 셀에 적용 할 수 있습니다.

비디오: Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

의 레이저 캡처 Microdissection Drosophila 주변 뉴런

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

연구

신경과학

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

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

잘라내기 -로드 : 망막 뉴런 사이 간격 접합 추적 커플링의 범위를 검토​​를위한 유용한 도구

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

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

차 해마 신경 세포의 칼슘 인산염 형질

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

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

신경 세포주에 신경 독성과 신경에 관여하는 분자 진학의 윤곽을하는 도구로 실시간 임피던스 기반의 세포 분석기

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 has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

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

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

모델로 후각 시스템은 축삭 성장 패턴 및 형태를 연구하는<em&gt; 생체</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

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

모터 엔드 플레이트 함께 근육 주사 : 직접 모터 뉴런에 셔틀 트레이서에 대한 최소 침습 접근

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

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

스트라이프 분석은 해리 해마 신경 세포를 사용하여 단백질 기판의 매력적인 또는 혐오감 활동을 연구하는

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

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hippocampal 슬라이스 문화 연구 Neuroprotection 및 종양 세포의 침입을 모델로

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

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

소 대형의 강력한 셀 모델로 기본 신경 하 Microsomes 관련 알파 synuclein 집계의 외 인 관리

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

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Microglia에 의해 항 체 중재 타우 정리 공부 세포 기반 분석 결과

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