여기에 설명 된 작업은 재향 군인의 업무의 미국학과 생물 의학 연구소 연구 개발 BX001686-1A1와 VA 재활 연구 및 개발에 의해 투자되었다.

세포 배양 1. 도금 및 유지 <ol><li> 따뜻한 둘 베코의 수정 이글 중간 (DMEM) 배지 (DMEM + 10 % FBS + 1 % 페니실린 / 스트렙토 마이신 / 네오 마이신) 37 ° C까지 </li><li> -80 ° C.에서 냉동 A12 세포의 주식을 확보 </li><li> 빠른 속도로 37 ° C의 물을 욕조에 세포를 해동; 한 번 해동, 부드럽게 피펫 세포는 75cm 2 배출 문화 플라스크에 전송하고 배양 배지 10 ㎖를 추가 할 수 있습니다. </li><li> 37 ° C에서 5 % CO 2 배양기에서 플라스크 하룻밤을 품어 미디어를 대기음 24 시간 후 신선한 용지로 교체합니다. 70-80% 합류는 (2 ~ 3 일 성장)을 얻을 때까지 세포 성장을 계속합니다. </li></ol> 2. 계수 및 도금 세포 <ol><li> 따뜻한 DMEM 37 ° C.에 1X-트립신-EDTA (에틸렌 다이아 민 테트라 초산) 솔루션 </li><li> 세포와 미디어를 대기음은 멸균 인산 완충 식염수 (PBS)로 2 회 세척한다. </li><li> 트립신 용액 500 μl를 추가하고 2-5 분 동안 37 ° C에서 알을 품다. </li><li> 분리스크레이퍼를 사용하여 미디어의 5 ML에있는 세포를 일시 중단 플라스크의 세포. 스트레이너를 통해 세포에 전달하고 깨끗한 50 ㎖ 튜브에 셀 스트레이너를 통해 전달합니다. 한 번 이상 스트레이너를 통해 세포를 통과 할 필요가 있습니다 만, 3 회 이상 반복하지 않습니다. </li><li> 미디어 100 μL의 최종 볼륨의 96 - 웰 분명 바닥 검은 색이나 흰색 벽으로 둘러싸인 플레이트에 6,000 세포 / 웰 시드 문화에 세포의 양을 결정하는 혈구를 사용하여 세포를 계산합니다. 24 시간 동안 37 ° C에서 5 % CO 2 판을 품어. </li></ol> 3. 세포 생존과 카스파 3 / 7 활동을 결정 <ol><li> 살균 5 ㎖의 유리제 바이알에 PA를 전송하고 100 % 디메틸 술폭 시드 (DMSO)에 용해. DMSO의 주식 PA 솔루션 1:10 희석하고 0.1 ㎜ PA의 최종 작업 농도를 얻기 위해 DMEM을 데워진에 추가합니다. 제어 매체의 경우, PA를 포함하는 미디어에 추가됩니다 DMSO의 동일한 농도를 추가합니다. </li><li> 물론 각각의 용지를 제거하고로 교체 50 μL 미디어 + / -PA. 37 ° C에서 5 % CO 2에서 2 시간 동안 접시를 품어 이어서 5 μL의 레사 주린 시약을 추가하고, 실온에서 10 분 동안 배양한다. </li><li> 세포 생존 능력을 측정하기 위해 멀티 모드 마이크로 플레이트 판독기, 형광 기록 (560 EX / 590 EM) 사용. 결과는 상대 형광 단위 (RFU)으로 표시하고 있습니다. </li><li> 같은 판에, 카스파 시약의 5​​5 μl를 추가하고 실온에서 2 시간 동안 배양한다. </li><li> 다시 카스파 3 / 7 활성을 측정하기 위해 마이크로 플레이트 리더, 기록 발광을 사용. 결과는 카스파 3 / 7 활동에 직접적으로 비례 발광으로, 상대의 발광 장치 (RLU)로 표현된다. </li><li> , 세포 수에 카스파 제 3 / 7 활동을 정상화 카스파 3 / 7 활동 (RLU)에 의해 세포 생존 능력 (RFU)를 나눕니다. </li></ol>

<ol>
	<li>Akil, L., Ahmad, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+obesity+and+cardiovascular+diseases+in+four+southern+states+and+Colorado.">Relationships between obesity and cardiovascular diseases in four southern states and Colorado.</a> J. Health Care Poor Underserved. 22, 61-72 (2011).</li><li>Posey, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+proinflammatory+lipid+accumulation,+inflammation,+and+insulin+resistance+in+rats+fed+a+high-fat+diet.">Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet.</a> Am. J. Physiol. Endocrinol. Metab. 296, 1003-1012 (2009).</li><li>Moraes, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fat+diet+induces+apoptosis+of+hypothalamic+neurons.">High-fat diet induces apoptosis of hypothalamic neurons.</a> PloS one. 4, (2009).</li><li>Mayer, C. M., Belsham, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+attenuates+insulin+signaling+and+induces+endoplasmic+reticulum+stress+and+apoptosis+in+hypothalamic+neurons:+rescue+of+resistance+and+apoptosis+through+adenosine+5'+monophosphate-activated+protein+kinase+activation.">Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation.</a> Endocrinology. 151, 576-585 (2010).</li><li>Benoit, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+acid+mediates+hypothalamic+insulin+resistance+by+altering+PKC-theta+subcellular+localization+in+rodents.">Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.</a> J. Clin. Invest. 119, 2577-2589 (2009).</li><li>Thaler, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+is+associated+with+hypothalamic+injury+in+rodents+and+humans.">Obesity is associated with hypothalamic injury in rodents and humans.</a> J. Clin. Invest. 122, 153-162 (2012).</li><li>Williams, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+dysfunction+in+obesity.">Hypothalamic dysfunction in obesity.</a> Proc. Nutr. Soc. 71, 521-533 (2012).</li><li>Belsham, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+phenotypic+array+of+hypothalamic+neuronal+cell+models+to+study+complex+neuroendocrine+disorders.">Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders.</a> Endocrinology. 145, 393-400 (2004).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+cell+viability+and+proliferation+in+hydrogel-encapsulated+system+by+resazurin+assay.">Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.</a> Appl. Biochem. Biotechnol. 162, 1996-2007 (2010).</li><li>Butterick, T. A., Nixon, J. P., Billington, C. J., Kotz, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orexin+A+decreases+lipid+peroxidation+and+apoptosis+in+a+novel+hypothalamic+cell+model.">Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model.</a> Neurosci. Lett. 524, 30-34 (2012).</li><li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+multiple+assay+endpoints+to+investigate+the+effects+of+incubation+time,+dose+of+toxin,+and+plating+density+in+cell-based+cytotoxicity+assays.+Assay+Drug+Dev.">Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev.</a> Technol. 2, 51-62 (2004).</li><li>Niles, A. L., Moravec, R. A., Riss, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+viability+and+cytotoxicity+testing+and+same-well+multi-parametric+combinations+for+high+throughput+screening.">In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening.</a> Curr. Chem. Genomics. 3, 33-41 (2009).</li><li>Steffen, W., Linck, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+immunoblot:+a+sensitive+technique+to+stain+proteins+and+detect+multiple+antigens+on+a+single+two-dimensional+replica.">Multiple immunoblot: a sensitive technique to stain proteins and detect multiple antigens on a single two-dimensional replica.</a> Electrophoresis. 10, 714-718 (1989).</li><li>Gingrich, J. C., Davis, D. R., Nguyen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+and+quantitation+of+proteins+on+western+blots+using+fluorescent+probes.">Multiplex detection and quantitation of proteins on western blots using fluorescent probes.</a> Biotechniques. 29, 636-642 (2000).</li><li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+multiple+assay+endpoints+to+investigate+the+effects+of+incubation+time,+dose+of+toxin,+and+plating+density+in+cell-based+cytotoxicity+assays.">Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays.</a> Assay Drug Dev. Technol. 2, 51-62 (2004).</li><li>Smyth, P. G., Berman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+apoptosis:+methods+for+elucidating+the+mechanism+of+apoptotic+cell+death+from+the+nervous+system.">Markers of apoptosis: methods for elucidating the mechanism of apoptotic cell death from the nervous system.</a> Biotechniques. 32, 648-650 (2002).</li><li>Lavrik, I. N., Golks, A., Krammer, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases:+pharmacological+manipulation+of+cell+death.">Caspases: pharmacological manipulation of cell death.</a> J. Clin. Invest. 115, 2665-2672 (2005).</li></ol>

저자는 공개 할 충돌이 없습니다.

멀티 플렉스 분석은 PCR 마이크로 어레이, 면역 및 기타 단백질 기반 검출 방법 13, 14 등 다양한 응용 프로그램에서 과학자들에 의해 사용 된 잘 받아 기술이다. 최근, 다중 분석이 점점 체외 플레이트 기반의 실험에 이용하고 세포 독성을 평가하는 정확한 방법으로 검증하고 12 생존 한되었다. 위의 프로토콜에서는, 우리는 PA의 반칙으로 A12 세포에서 카스파 제 3 / 7 활동과 세...

팔 미트 산 (PA) 등의 포화 지방산이 풍부한 다이어트는 비만과 심장 혈관 질환과 당뇨병 1, 2 등 다른 동반 질환에 연결되어있다. 고지방 사료 또한 산화 스트레스, 아폽토시스 및 시상 하부 신경 변성, 식욕 및 에너지 비용 3-7의 중요한 레귤레이터를 증가하는 것으로 나타났다. 높은 지방 다이어트 노출 시상 하부 조절 장애를 유발 통해 메커니즘을 이해하는 것은 비만에 대한 약물 치료의 발전을 위해 이렇게 중요합니다. 그러나,식이 지방이 신경 세포의 기능에 영향을 미치는 통해 세포 메커니즘은 불분명하다. 이러한 PA 등의 지방이 시상 하부의 세포 사멸 경로의 발병을 유발하는 방법의 더 나은 이해는이 목표를 향해 필요한 첫 번째 단계입니다. 이 문서의 목적은 시간의 연구에 사용하기 위해 개발 PA 노출에 신경 반응의 시험관 테스트를위한 다중 분석을 설명하는 것입니다ypothalamic 신경 퇴행. 우리는 PA 8 산화 공격 후 (A12)의 이용 차별화 된 불후의 성인 마우스 시상 하부 세포주에서 세포의 총 수 당 3 / 7 활동 카스파를 측정하기위한 시험 관내 96 - 웰 형식의 멀티 플렉스 분석에 대한 자세한 설명을 제공한다. 간단히, 세포 생존 능력은 레사 주린 기반 분석을 통해 PA의 반칙으로 결정된다. 레사 주린은 대사 활성 세포에있는 효소의 감소를 겪는 세포 투과성 화합물, 생각 프로세스는 미토콘드리아 (9)를 통해 발생합니다. 생존 세포는 지속적으로 생존 세포의 수에 비례하여 형광 신호를 생성하는, 레조 루핀 (resorufin) 레사 주린하는 변환. 카스파 제 3 / 7 활성은 다음 DEVD lumogenic 기반 분석을 사용하여 분석된다. DEVD는 카스파 제 -3에 의해 절단 아미노산 서열 (ASP-글루 - 발-ASP)이다. 이 서열은 세포 내 카스파 3 / 7의 활성화 및 후속 절단시 lumogenic 물질에 결합되면DEVD 기판으로, 발광 제품이 해제됩니다. 이 반응은 카스파 제 활성에 따라서 세포 자멸사의 유도에 비례한다. 사균은 카스파 제를 생산할 수 없기 때문에, 카스파 제 3 / 7 활성은 천연 과도로이고; 따라서 분석은 세포 스트레스의 효과에 따라, 4 시간 후 - 도전 30 분 사이에 완료되어야한다. 세포 생존율은 카스파 제 3 / 7 활성에 반비례하고 세포 사멸의 메카니즘을 결정하는데 사용될 수있다. 예를 들어,이 방법은 이전에 오렉신이 처리에 의해 영향을받는 메커니즘이 산화 손상 (10)에 대한 보호에 중요하다는 것을 시사 과산화수소 도전 시상 세포에서 세포 사멸을 감소 펩타이드 호르몬이 전처리를 표시하는 데 사용되었다. 그것은 그들이 분석 시약을 줄이기 위해 미토콘드리아 활성에 의존하는 이러한 어 세이는 세포주 및 조직 의존하는 것이 중요하다. 이 프로토콜은 성인 마우스의 시상 하부 (A12) 셀에 최적화되어 있습니다; 그러나,기재된 방법은 유사한 연구의 범위에 맞도록 변경 될 수있다. 단일 문화권에서 다중화 분석법 확실히 여러 가지 이유로 개별 분석을 수행하는 기존의 방법보다 이점을 제공한다. 시간, 세포 샘플 및 배양 시약을 저장하는 것 외에도, 다중화 분석법은 또한, 세포의 생존 및 사망의 지식을 제공 내부 제어를 제공하고, 반복적 인 실험 11, 12에 대한 필요성을 제거 할 수있다. 다른 방법은 96 - 웰 형식을 사용하여 특히, 신뢰할 수있는 분석이다 서양 말이나 ELISA를,에 의존하지만, 비용과 시간이 많이 소요 (1-2 일간)입니다있다. 이 다중화 분석을위한 세포 배양에 소요되는 시간을 제외하고, 전체 시간은 3 시간 미만이다. 이 프로토콜은 A12 세포와 함께 사용하기 위해 최적화되어 있지만 셀의 무결성에 영향을 미칠 수있는 요인 염두에 유지하면서, 다른 모델에서 사용하기 위해 변경 될 수있다. 제지이 프로토콜은 일련의 실험을 위해 일하면 마이닝은 샘플이나 실험 조건의 수에 계획된 다운 스트림 실험에 따라 달라집니다.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Adult Mouse Hypothalamus Cell Line mHypoA-1/2&#160;</td>
			<td style="width:137px;">Cellutions Biosystems Inc.</td>
			<td style="width:151px;">CLU172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Fetal bovine serum&#160;</td>
			<td style="width:137px;">PAA Labs</td>
			<td style="width:151px;">A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Penicillin/Streptomycin</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Palmitic Acid</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethy sulfoxide&#160;</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;"> 
			PrestoBlue Cell Viability Reagent</td>
			<td style="width:137px;">Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Caspase-Glo 3/7 Assay Systems</td>
			<td style="width:137px;">Promega</td>
			<td style="width:151px;">G8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Equipment</td>
			<td style="width:137px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:268px;">96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td>
			<td style="width:137px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">165305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">SpectraMax M5 Multi-Mode Microplate</td>
			<td style="width:137px;">Molecular Devices</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model

복잡한 세포 메커니즘의 연구에서 분석을 다중화하는 능력은, 반복적 인 실험의 필요성을 없애 내부 제어를 제공하고, 비용 및 시약에 폐기물을 감소시킨다. 여기에서 우리는 96 - 웰에서 생존 세포 수와 카스파 제 3 / 7 활성을 측정하기 위해 기반 분석법을 형광체 및 발광을 모두 사용하여, 시험 관내 시상 모델의 팔 미트 산 (PA)의 반칙 아폽토시스를 평가하기 위해 멀티 플렉스 분석의 최적화를 설명 마이크로 플레이트 형식입니다. PA의 반칙으로, 가능한 세포는 레사 주린 기반의 형광 분석에 의해 결정되었다. 카스파 제 3 / 7 활성은 luminogenic 기판 DEVD 및 세포 수로 정규화를 사용하여 측정 한 다음이었다. 이 다중 분석법은 포화 지방산 도전으로 사멸 자극, 다음 카스파 제 활성의 변화를 결정하기위한 유용한 기술이다. 포화 지방산 PA는 잠재적 importa을 나타내는 시상 산화 스트레스 및 세포 사멸을 증가시킬 수있다예컨대 그와 같은 분석법의 NCE은 포화 지방산과의 연결 기능 사이의 관계를 연구하는 기재.

프로토콜은 위 세포 죽음의 메커니즘을 결정하는 다중 두 개의 분석 결과를 설명합니다. 그림 1 세포 생존 및 카스파 제 3 / 7 활동을 결정하는 프로토콜에 대한 개요를 보여줍니다. 카스파의 활동은 크게 2 시간 배양 (그림 2A 표 1) 후 PA에 도전 세포에서 증가했다. 세포막의 무결성의 손실은 PA (그림 2B)에 2 시간 노출 후 세포에서 명백한 세포 사멸 유도와 관련?...

Watch this Scientific Journal Video about Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model at JoVE.com

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

멀티 플렉스 분석은 기본 세포 메커니즘에 대한 유익한 정보를 제공하고 시약 및 불필요한 반복 실험의 낭비를 제거 할 수 있습니다. 우리는 팔 미트 산으로 산화 반칙으로 체외 시상 하부 모델에서 세포 생존을 결정하기 위해, 형광 및 발광 기반의 방법을 사용하여, 여기에 멀티 플렉스 카스파 3 / 7 활동 분석에 대해 설명합니다.

시상 하부 세포 모델에서 세포 사멸을 결정하는 카스파 다중 분석의 사용

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

use-caspase-multiplexing-assay-to-determine-apoptosis-hypothalamic

Research

JoVE Journal

Neuroscience

14.9K 조회수.  Minneapolis Veterans Affairs Health Care System.  멀티 플렉스 분석은 기본 세포 메커니즘에 대한 유익한 정보를 제공하고 시약 및 불필요한 반복 실험의 낭비를 제거 할 수 있습니다. 우리는 팔 미트 산으로 산화 반칙으로 체외 시상 하부 모델에서 세포 생존을 결정하기 위해, 형광 및 발광 기반의 방법을 사용하여, 여기에 멀티 플렉스 카스파 3 / 7 활동 분석에 대해 설명합니다. 

비디오: Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

시신경의 절개 : 중추 신경계에서 신경 세포의 Apoptosis 성인의 모델

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

연구

신경과학

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

세포 어세를 형성 신경 콜로니 : 신경 전구 세포로부터 타고난 신경 줄기 세포를 차별 어세이

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

모니터링 죽습 Caspase - 3 활동 및 칼륨 - 유도 막 탈분극 다음 라이브 세포 이미징 및 Cleaveable 형광 염색 기판을 사용하여 불후의 Oligodendroglial 세포의 Apoptosis

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

초점 방사선 억제와 성인 시상 하부의 신경 발생의 기능 심문

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

성인 Zebrafish의 망막 재생 연구에있는 사용을위한 비발 한 가벼운 손상 패러다임

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

나노 입자 신경 독성을 결정하는 대리 바이오 센서로 미세 아교 세포

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

태아 알코올 스펙트럼 장애의 쥐 모델에서 Hippocampal 기능 장애를 평가 하기 위해 추적 Eyeblink 클래식 컨디셔닝의 사용

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

라이브 셀 측정의 Odorant 수용 체 활성화를 사용 하 여 분석 결과 실시간 캠프

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

코카인 사용 장애의 쥐 모델에서 큐 반응성을 측정 하기 위한 프로토콜

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

최소한의 척수 상해의 마우스 모델에서 내 인 성 신경 줄기 세포 활성화 평가 Neurosphere 분석 결과

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...