우리는이 원고의 중요한 읽기위한 마츠 실험실의 구성원 감사합니다.

1. 절차 : 절차는 삼일 총 시간이 지남에 따라 완성된 세 부분으로 구성되어 전송 세포 transfection과 immunocytochemistry, 하루에 실시 한 걸음. 처음 두 일 동안 전송 및 세포의 transfection은 층류 챔버에 무균 상태에서 실시해야합니다. 1 일 : 표면 표현 분석에 대한 HEK293T 세포의 양도. <ol><li> 전송 매체 (M10) : 믹스 10 % (부피가) 무균 상태에서 태아 소 혈청과 보충 최소 필수적인 매체. </li><li> 설정하는 데 필요한 접시의 숫자에 따라, 100mm 문화 판에서 원하는 confluency에 세포를 성장. 자란 또는 스파스 세포를 피하로 전송하는 세포의 분율을 결정하는 것이 중요합니다 : 예를 들어, 100 % 합류 100mm 요리에서, 하나는의 30 % confluency를 구하는 35mm 요리로 약 3 %를 양도할 수 있습니다 후자. </li><li> 이전 멸균 층류 챔버, 코트 22, 세포를 전송 멸균 폴리 D 라이신 (1mg / ML) 각 35mm의 세포 배양 접시에서 개최 한 도금 전지 코팅 측면까지 함께 X 22mm 유리 커버 풀렸는데 (그림 1 ). 십분 - 5 건조에 대한 해결책을 허용합니다. 원하는 경우이 간격 동안 판상 챔버의 자외선 소스, 커버 전표를 소독에 설정되어있을 수 있습니다. 폴리 라이신은 D heterologous 세포 커버 전표에 충실하는 데 도움이됩니다. </li><li> 세포를 전송하려면, 100mm 접시에있는 기존의 미디어를 대기음, 조심스럽게 8 ML 멸균 PBS, 대기음 PBS를 추가하여 씻어 3 ML에게 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)를 사용하여 세포를 trypsinize, 세포 접시에서 분리하면에 의해 추가로 효소 반응을 차단 5 ML의 M10을 추가. 세포를 씹다하고 살균 원뿔 관에 필요한 볼륨을 전송, 펠릿 세포 5 분 200g에 원심 분리기. 세포의 펠렛은 그대로두고, 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)를 포함 뜨는을 대기음. 35mm 요리에 세포를 전송하려면 각 접시 1 ML M10를 추가하고 세포를 떼어 놓다을 씹다. 각 접시에 resuspended 세포 1 ML을 추가합니다. 37 밤새 품어 세포 ° C, 5 % CO 2. <img src="/files/ftp_upload/2405/2405fig1.jpg" alt="그림 1" /> 그림 1. 코트 22 폴리 라이신 D 각 35mm 접시에 놓으 코팅 측면을 함께 X 22mm의 유리 coverslips. </li></ol> 일 2 : HEK293T 세포의 Transfection. <ol><li> 전송 후 18~24시간은 전지 assayed 수 수용체의 transfection에 대한 준비가되어 있습니다. 수용체 4 언급한 Qiagen Miniprep 키트 다음과 같은 프로토콜을 사용하여 세균의 문화에서 준비 포유류의 표현 벡터 PCI에서 복제 RTP1S을 사용합니다. N 말기 immunostaining을 촉진 20 아미노산 긴 rhodopsin 에피토프가 추가됩니다 수용체를 사용하십시오. </li><li> 수용체 구조, RTP1S 구조 250 NG와 녹색 형광 단백질 각각 transfection 50 NG의 1000ng를 사용하십시오. 제조 업체의 설명서를 따라 같은 Lipofectamine 2000을 사용하여 transfection을 수행합니다. </li></ol> 3 일 : 세포 표면 수용체 자국을 시각화하기 위해 Immunocytochemistry. <ol><li> 시작하기 전에하는 얼룩 및 솔루션을 씻고 준비를 4 ° C. 그들을 냉각 매체 얼룩 : 최소 필수 중간, 45 ML, 태아 소 혈청, 5 ML, HEPES (1M), 500 μL (최종 농도 10mM), 나트륨 Azide (1.5M), 500 μL (최종 농도 15mM). ; HEPES (1M), 5 ML (최종 농도 10mM), 나트륨 Azide (1.5M), 5 ML (최종 농도 15mM) HBSS, 500 ML : 솔루션을 씻으십시오. 두 솔루션은 재사용 4 ° C에 저장된 수 있습니다. 얼룩 매체 100 배에 필요한 항체를 diluting하여 항체 솔루션을 준비합니다. </li><li> 24 시간 게시 transfection에 대한 odorant 수용체에 대한 표면 얼룩에 대한 immunochemistry를 시작합니다. 라이브 세포 얼룩, 4 냉각해야 최대 실험 설정을 수행하려면 ° C : 입력 대형 얼음 포함되어 등도 가능한 한 그것을 만들기 위해 얼음의 표면을 두드려, 얼음에 트레이를 배치 70 %를를 스프레이 에탄올. 부드러운 표면을 얻기 위해, parafilm의 조각 (트레이의 길이보다 작은)와 에탄올 물을 뿌리면 트레이에 부착 그것을 잘라. 유리 커버가 핀셋을 사용하여 parafilm에 transfected 세포, 한 번에 하나, 세포 측면을 포함하는 전표 전송합니다. 레이어 각각의 커버 슬립 (그림 2)의 일각에서 조심스럽게 압출, 100 μL 차가운 차 얼룩 솔루션 전표. 겹겹이 항체 솔루션으로 모든 표지 풀렸는데 후, 공기 솔루션의 최대 건조 방지하기 위해 트레이를 다룹니다. 45-60분에 대한 기본 부화를 수행합니다. </li><li> 기본 부화 후, 신속하게 미디어를 포함, 원래 35mm 문화 요리 커버 전표를 전송할 수 있습니다. 미디어를 기음과 커버 슬립의 세포를 잃는 것을 방지하기 위해 접시의 가장자리에서 천천히 두 ML 차게 씻어 솔루션을 추가합니다. 세차 솔루션을 기음 세 세척을 완료하는 두 번 반복합니다. 세 번째 세척의 끝에서 씻어 솔루션을 대기음하지 않습니다. </li><li> 이차 항체 soluti 준비얼룩 미디어에서와 같은 트레이에 새로 부착된 parafilm 레이어로 세척 솔루션에서 커버 전표를 전송할 수 있습니다. 다시 부드럽게 레이어 기본 부화 같은 코너에서 각 커버 슬립을하는 항체 용액 100 μL를 돌출. 30 차 배양을 수행 - 45 분 거리에 있으며 원래 35mm 요리에서 세척 솔루션에 다시 전송할 수 있습니다. 한 번 더, 뚜껑이 설명한대로 세 번 전표 씻으십시오. </li><li> 마지막으로 씻어에서 솔루션을 씻어 2 ML 1퍼센트 차게 paraformaldehyde를 추가 대기음. 15 분 동안 얼음에 차게 1 % paraformaldehyde 용액에 세포를 수정. </li><li> 깨끗한 현미경 유리에 커버 전표를 마운트 다운 세포 측면으로 장착 시약의 Mowiol를 사용하고 신중하게 모든 공기 방울 (그림 3)을 제외 슬라이드. 일단 Mowiol 공기 증발, 표면에 증류수를 추가하고 즉시 aspirating하여 부드럽게 커버 전표를 씻으십시오. <img src="/files/ftp_upload/2405/2405fig2.jpg" alt="그림 2" /> 그림 2. 층 100 μL 차가운 차 얼룩 솔루션으로 각 coverslip. <img src="/files/ftp_upload/2405/2405fig3.jpg" alt="그림 3" /> 그림 3. 깨끗한 유리 현미경 아래에서 셀 사이드로 coverslips을 마운트 Mowiol 장착 시약을 사용하여 슬라이드. </li></ol> 2. 대표 결과 : 라이브 세포 얼룩은 보호자 (수용체 운반 단백질 RTP1S이 경우 odorant 수용체 Olfr62에서) (그림 4)와 수용체의 transfection에 세포의 표면에 단백질을 시각화 한 수 있습니다. 수용체의 세포 표면 얼룩은 얼룩의 작은 반점이있는 패턴 (그림 4B, 삽입된 페이지)에 의해 특징입니다. 얼룩은 어렵게 관찰자가 소음에서 실제 표면 얼룩을 구분하는 만들기, 부적절하게 높은 노이즈가 발생할 수 있습니다 진행. <img src="/files/ftp_upload/2405/2405fig4.jpg" alt="그림 4" /> 보호자와 수용체의 그림 4. Transfection 살 세포 얼룩을 사용하여 세포의 표면에 단백질의 시각화를위한 수 있습니다. RTP1S의 보호자 (B)와 odorant의 혼자 수용체 Olfr62 (A)와 Olfr62의 transfection의 Transfection. GFP 표현 수준 transfection 제어 (A &#39;와 B&#39;) 역할을합니다.

<ol>
	<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65, 175-187 (1991).</li><li>Malnic, B., Hirono, J., Sato, T., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+receptor+codes+for+odors.">Combinatorial receptor codes for odors.</a> Cell. 96, 713-723 (1999).</li><li>Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119, 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nat Protoc. 3, 1402-1413 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

각 단계는 두드러진과 독특한 표면 얼룩을 보장하기 위해 신중하게 진행되어야합니다. 전체 얼룩 과정 (얼음) 추위에 실시해야하며 커버 전표가 마련하고있는 트레이는 세포가 살아있을 수 있도록 사용하기 전에 냉각해야합니다. 또한 고정은 표면 얼룩 프로세스의 끝에 이루어집니다. 이것은 고정은 기본 및 보조 항체로 세포막을 permeabilize에 얼룩 앞에 내부 얼룩과 선명한 대조이다. 공기 커버 ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cover slip 22 X 22 mm (#1)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>72200-11</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>&nbsp;</td>
<tr>
<td>Minimal essential medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4655</td>
<td>With L glutamate, Earle&#x2019;s salt and bicarbonate</td>
</tr>
<tr>
<td>Mowiol</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>475904</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>EMS</td>
<td>19208</td>
<td>&nbsp;</td>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) 1X </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>14025</td>
<td>With calcium chloride and magnesium chloride</td>
</tr>
<tr>
<td>HEPES Buffer Solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Saline (PBS)</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-040-CV</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Poly D Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Azide</td>
<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0299-1</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dish 35 X 10 mm</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353801</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>0.05%</td>
</tr>		</tbody>
		</table>
		</div>

assaying surface expression of chemosensory receptors in heterologous cells

냄새의 생생한 세상은 후각의 감각에 의해 인식됩니다. 생쥐의 후각은 휘발성 odorant 분자 바인딩하는 postulated 및 G 단백질과 결합하여 Gαolf 세포내 신호에 세포 신호를 변환하는 1200에 대한 G 단백질 결합 수용체의 (GPCRs) 1 레파토리에 의해 중재됩니다. 수용체에 odorants의 바인딩하면 하나 odorant 여러 수용체를 묶어 수 있습니다 한 수용체가도 2 다양한 여러 odorants를 바인딩 수있는 조합 규칙을 따를 생각입니다. 생화학, 신호 및 리간드 바인딩 연구 편리 heterologous 세포를 사용하는 대부분의 GPCRs에 대한 실시되었습니다. transfection에 그들은 표면에 수출하는 데 실패 때문에 그러나, odorant 수용체의 연구 heterologous 세포의 사용은 오랫동안 배제되었다. 사이토 외 가족 보호자들이 공동으로 함께 3 transfected heterologous 세포의 표면에 트래픽 odorant 수용체에 보호자로 후각 감각 뉴런과 행동의 강화된 표현을 보여주 단백질을 운반 단일 막 통과 수용체 (RTP)을 증명하고있다. 수용체는 세포 라인의 강력한 표면 표현을 보여주는 경우 heterologous 세포를 이용하여 수용체에 대한 생화 학적 assays을 수행하는 하나 먼저 확인해야합니다. 이것은 찬란 독점적으로 세포외 도메인의 세포외 도메인이나 태그를 레이블에 얼룩 라이브 셀 다음에 보호자의 RTP1S와 overexpressing 수용체에 의해 assayed 수 있습니다. 우리가 편리하게 HEK293T 세포의 표면에 odorant 수용체를 감지하는 데 사용할 수있는 라이브 세포 얼룩을 수행하는 프로토콜을 보여줍니다. 또한, 그것은 또한 다른 chemosensory 수용체 또는 GPCRs의 표면 표현 분석하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells at JoVE.com

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

우리가 편리하게 HEK293T 세포의 표면에 odorant 수용체를 감지하는 데 사용할 수있는 라이브 세포 얼룩을 수행하는 프로토콜을 보여줍니다. 또한, 그것은 또한 다른 chemosensory 수용체 또는 GPCRs의 표면 표현 분석하는 데 사용할 수 있습니다.

Heterologous 셀에 Chemosensory 수용체의 표면 표현을 시금

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

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Research

JoVE Journal

Neuroscience

10.8K 조회수.  Duke University. 우리가 편리하게 HEK293T 세포의 표면에 odorant 수용체를 감지하는 데 사용할 수있는 라이브 세포 얼룩을 수행하는 프로토콜을 보여줍니다. 또한, 그것은 또한 다른 chemosensory 수용체 또는 GPCRs의 표면 표현 분석하는 데 사용할 수 있습니다.

비디오: Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Circadian 리듬을 연구 및 매개 변수 잠을 전위의 활동 시금 Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

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신경과학

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

에 시금 운동력이있는 사람, 학습, 및 메모리 적자 Drosophila</emNeurodegeneration의> 모델

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

척수 전기 생리학 II : 세포 흡입 전극 제작

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

설치류 또는 인간의 네이절 강한 줄기 세포를 분리

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

혜성 분석을 사용하여 Hippocampal 뉴런의 DNA 손상을 시금

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

게 사지에서 고유 감각과 긴장 수용체 : 학생 실험실 연습

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

신경 발사 환율의 조작에 의해 성상 세포 수용체의 가소성을 유도

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Biotinylation을 사용하여 기본 Astrocyte 문화에서 단백질의 세포 표면 표현 및 Endocytic 속도를 결정

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...