저자는이 방법의 초기 기반에 대한 존 울포드을 인정하고 싶습니다, TGK는 NIH GM057483와 AI076245 지원하고 PRC는 GM77073에 의해 지원됩니다.

1. 7~47%의 자당의 그라디언트의 준비 <ol><li> 그라디언트가 지속 될 수 있도록 사용하기 전에 자당의 그라디언트 한 하루를 준비합니다. 7 % 살균하고 4 50mM NH 4 Club 호텔을 포함 47%의 자당 솔루션, 50mM 트리스 - 아세테이트 산도 7.0과 장총 MgCl 2와 저장 ° C 1. </li><li> 6 그라디언트는 다음과 자당의 농도 각각 48 ML을 할 수있는 7퍼센트 47 %의 자당 주식 솔루션을 혼합하십시오. 1mM의 최종 농도 각 자당 솔루션 DTT를 (1M 주식)을 추가합니다. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 최종 농도 </td><td> 7 % 자당 </td><td> 47%의 자당 </td></tr><tr><td> 7% </td><td> 48 ML </td><td> 0 </td></tr><tr><td> 17% </td><td> 36 ML </td><td> 12 ML </td></tr><tr><td> 27% </td><td> 24 ML </td><td> 24 ML </td></tr><tr><td> 37% </td><td> 12 ML </td><td> 36 ML </td></tr><tr><td> 47% </td><td> 0 </td><td> 48 ML </td></tr></tbody></table></li><li> 그라디언트를 부어하기 위해 확보하고 Parafilm와 함께 밀봉하여 20 ML의 주사기에 파스퇴르 피펫을 첨부하거나 긴 바늘을 사용합니다. 1 X 3.5 &quot;polyallomer의 초원 심 분리기 튜브의 바닥에 7 % 자당 7 ML을 추가합니다. 다음 0.3 이상의 튜브 하단 파스퇴르 피펫 팁을 배치하고 더 빨리 pipetting 없다하여 7 % 용액에 따라 17 % 자당 솔루션 7 ML을 추가 7 ML 27%의 각 37% 47 %의 자당 솔루션 ML / 초. 반복. 당신은 혼합을 최소화는 ° C 하룻밤 로터, 병과 함께 4. 스토어 그라디언트를 발생했음을 나타내는 레이어 사이의 명확한 라인을 준수해야합니다 그리고 샘플을 수확하는 데 사용됩니다 튜브. </li></ol> 2. 효모 추출물의 준비 <ol><li> 0.8-1.0의 OD 600 효모 문화의 125 ML 성장. 100 μg / ML의 최종 농도에 문화 cycloheximide를 추가하고 30 흔들어 ° C를 15 분 동안 계속. </li><li> 한편, 80 μg / ML cycloheximide, 200 μg / ML 헤파린, 0.2 % DEPC, 10 MM 트리스 - HCL 산도 7.5, 0.1M NaCl, 30mM MgCl 2가 포함된 용해 버퍼를 준비합니다. 이 시점부터 얼음에있는 모든 보관하십시오. </li><li> 250 ML의 원심 분리기 병에 문화를 붓고 얼음과 함께 입력합니다. 4 5 분 ° C. 8000 XG (SLA - 1500 로터에 7250 RPM)에서 원심 분리기 얼음 차가운 용해 완충액 15 ML의 폴리 프로필렌 튜브로 전송 10 ML과 함께 한 번 펠렛 씻으십시오. 4 3 분 ° C.에 대한 5900 XG (SLA - 600TC 로터에 ~ 6,000 RPM)에서 원심 분리기 1.5mL 튜브에 용해 버퍼, 이전 0.5 ML의 펠렛을 Resuspend 및 냉장, 구운 산성 - 세탁 유리 구슬의 0.5 ML를 추가합니다. 와동 각 간격 사이에 30 초에 대한 얼음 튜브를 냉각 사 30 초 간격에 대한 세포. 1.5 ML 튜브에 용해 버퍼의 또 다른 0.5 ML를 추가합니다. 4 10 분 ° C.에 대한 microcentrifuge의 최대 속도 (Eppendorf 5417R의 microcentrifuge에서 14,000 RPM)에서 원심 분리기 새로운 1.5 ML의 microcentrifuge 관에 뜨는 전송합니다. OD 260 / OD 280 비율 (아래의 단계 3.1 참조)를 확인하기 위해 별도의 튜브를 10 μl를 제거합니다. -80에서 보관 추출물 ° C. </li></ol> 3. 포유 동물 세포에서 추출물의 준비 <ol><li> 자기편 세포 들어, ~ 70 % 합류로 100mm 요리 성장. 당신은 11 ML 기울기 당 하나의 100mm 요리 (전형적인 수율은 ~ 20 OD 260 단위입니다)이 필요합니다. 크거나 작은 기울기 크기에 대한 선형 금액을 크기. 비 점착 성의 세포의 경우, 약 1 X 10 7 세포는 11 ML 기울기마다 필요합니다. </li><li> 이전 용해에, 100 μg / ML에 cycloheximide를 추가합니다. 37 ° C 15 분 알을 품다. 얼음 접시를 전송하고 얼음 차가운 PBS로 두 번 세포를 씻어. 모든 미래의 단계 0-4 ° C.에서 진행 반드시 </li><li> 흡인에 의해 PBS의 모든 흔적을 제거 (접시는 얼음의 직각 침대에 유출하자) 1 ML의 용해 버퍼, 긁어 및 Pre - 차게 1.5 ML 튜브로 전송을 추가합니다. 용해 버퍼 구성 요소는 세포 유형과 성장 조건 최적화해야합니다. 좋은 시작 버퍼는 20 MM HEPES - 코, 산도 7.4, 15 MM MgCl 2, 200 MM KCl, 1 % 트리톤 될 X - 100 (V / V), 100 μg / ML cycloheximide, 2 MM DTT, 그리고 / 1 MG ML의 헤파린. 주요 변수는 마그네슘과 칼륨 염화물뿐만 아니라 cytoskeletal 또는 막 바운드 polysom​​es의 추출을 향상시키기 위해 포함 시키거나 비 이온 세제의 배제의 농도입니다. 두 번 27 게이지 주사 바늘을 통과 또는 B 타입 유봉을 사용하여 다운스의 균질의 50-10 스트로크에 의해 세포를 Lyse. 주사기 또는 균질 화기는 사용하기 전에 미리 차게해야합니다. </li><li> 냉장 microcentrifuge로 5 분 14,000 XG에 스핀. 자당 기울기 (포유 동물 세포에 대해 이것이 X - 100과 헤파린 튼 부족 용해 버퍼에서 만들지만 별도로 1.2와 1.3에 설명된) 또는 나중에 사용하기 위해 액체 질소의 플래시 프리즈 신선한 lysate를 전송합니다. </li></ol&gt; 4. 그라디언트의 원심 분리 <ol><li> 얼음 polyribosome lysates를 녹여. 물 1 ML에 예약된 10 μL 샘플을 추가하고 분광 광도계로 OD 260 / OD 280 비율을 읽어 lysate에서 핵산의 농도를 결정합니다. 설명한대로 성장 효모 문화 일반적인 수확량은 50 OD 단위입니다. </li><li> 그라디언트의 표면 근처 튜브의 벽, 부드럽게 레이어 그라디언트 위에 lysate 25 OD 260 단위에 대한 배치 끝에있는 피펫을 사용합니다. 에 대한 lysate의 35 ML 기울기 500 μl는 일반적으로로드됩니다. 용해 버퍼는 모든 그라디언트가 동일한 볼륨 로드된 보장하는 데 사용할 수 있습니다. 재료의 낮은 금액은 더 나은 해상도를 얻을 수 있습니다. 포셉 사용하여 신중하게 스윙 버킷 로터의 버킷에 그라디언트를 낮춥니다. </li><li> 4 4 시간 100,000 XG (Surespin 630 로터에 23,000 RPM)에서 그라디언트를 원심 ° C. 이 프로토콜은 작은 볼륨 그라디언트에 대해 적용할 수 있습니다. </li></ol> 5. 데이터 및 분수의 수집 <ol><li> 전에 4 시간의 스핀의 완료 약 30 분 모델 184 튜브 찌르는에 바늘을 부착합니다. 온라인 Isco 모델에 펜을 부착 UA - 5 흡광도 / 형광 모니터. 흡광도에 전원이 안정 기준이 사전 분석 샘플에 접근할 수 있도록 모니터링합니다. polysom​​e 프로파일 전자 인수 쉽게 차트 레코더 DI - 148U 데이터 수집 장치 (DATAQ 계측기)를 부착하여 얻을 수 있습니다. 프로필은 다음 실시간으로 수집 및 동반 WinDaq 소프트웨어를 사용하여 나중에 해설을 위해 저장할 수 있습니다. </li><li> Fluorinert 역류, 빠른 설정을 사용 50mL와 주사기 펌프를 채우십시오. 현재에는 기포가 없는지 확인합니다. 주사기에서 튜브 찌르는에 호스를 연결하고 간략하게 어떤 공기 방울의 호스를 취소하고 앞으로 방향 3 ML / 분 Fluorinert의 흐름을 켭니다. </li><li> 샘플을 수집하는 흐름 세포의 끝에 호스에 따라 분수를 수집 도망하면 튜브의 폐기물 비커 또는 시리즈를 놓습니다. </li><li> 조심스럽게 원심 회전 날개에서 자당 기울기를 포함하는 polyallomer의 원심 튜브를 제거하고 (교란 그라디언트를 피하기 위해 빈 튜브와 함께 얼음에있는 우물을 미리 양식) 얼음에 그들을 놓으십시오. </li><li> 세포 추출물을 분석하려면, 튜브 찌르는에 그라디언트 튜브를 놓습니다. 콘센트에 튜브의 상단을 연결 및 보안. 바늘이 튜브의 바닥을 피어 있도록 원심 튜브에 아래의 단계를 올립니다. 바늘이 원심 분리기 튜브의 아래쪽으로 돌출되면, 6mL/min에서 앞으로 방향으로 Fluorinert의 흐름을 시작한다. </li><li> Fluorinert가 튜브로 흐르는 시작되면, 온라인 흡광도 모니터에 바늘의 움직임을 모니터링합니다. 일단 바늘 60의 속도 설정으로 차트의 움직임을 설정, 이동하기 시작합니다. 모니터는 40 ribosomal subunit 감지 및 polyribosome 단지까지 계속이어서 자유 자재로 시작 OD 254를 기록합니다. 이 RNA 또는 단백질 분석을위한 분수를 수집하는 경우, 일반적으로 0.2 분 분수 (1 ML) 촬영입니다. RNA의 분석을 위해, 분수가 추출의 기본 방법에 따라 Trizol (Invitrogen), 페놀 또는 SDS / proteinase K를 포함하는 튜브에 직접 수집해야합니다. </li><li> polyribosome 프로필의 끝에 도달했습니다 때, 원심 튜브에 Fluorinert의 흐름을 해제하고 흡광도 / 형광 모니터의 차트 운동을 중지합니다. </li><li> 주사기에 튜브에서 자당을 그릴하지 않도록하고 빠른 속도로 반대 방향으로 흐름을 시작하여 주사기로 원심 튜브에서 Fluorinert을 철회. 튜브 찌르는에서 돌려서 원심 관은 바늘을 낮게하고, 튜브를 제거합니다. </li><li> 반복 각 자당 기울기에 대해 5.8을 통해 5.5 단계를 반복합니다. </li><li> 모든 자당의 그라디언트가 분석되었습니다되면, 주사기로 다시 호스에서 Fluorinert을 철회. 상단에있는 폐기물 호스 포함하여 fractionator의 각 부분을 (튜브 찌르는 및 흐름 세포)를 제거하고 물로 잘 씻어. </li></ol> 6. 대표 결과 <img src="/files/ftp_upload/1948/1948fig1.jpg" alt="그림 1" /> 그림 1. ribosome 추출 대표 추적은 cycloheximide의 존재에 효모 변형 BY4741 (매트 his3 Δ Δ 1 leu2 0 met15 Δ 0 ura3 Δ 0)에서 준비했습니다. ribosomal 추출물 7 47%의 자당 기울기를 이용 fractionated되었고 UV 검출기에 연결된 펌프 주사기 장치를 사용하여 분석했다. polyribosomes와 80, 60 및 40 변이를 포함하는 봉우리가 표시됩니다.

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	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

polyribosome 프로필에서 얻은 정보는 세포의 translational 상태에 귀중한 통찰력을 제공할 수 있습니다. 또한, ribosomal subunits 자체의 조립 상태가 2 결정하실 수 있습니다. 예를 들어 halfmers 또는 80 년대와 프로필에 오른쪽으로 약간 어깨와 큰 봉우리의 존재는 바운드 40가 가입 60을 기다리는 subunit subunit 나타냅니다. 어떠한 추가 cycloheximide이나 신장의 다른 억제제의 부재에서 실험을 수행하는 것은 신장이 3 변경할지 여부를 나타냅니다 도망의 속도의 분석을 위해 수 있습니다. 분수 자체는 북부 또는 서양 분수의 모래 바닥으로 ribosomal subpopulations있는 특정 mRNA 또는 단백질의 협회의 후속 결정을위한 시약의 풍부한 원천입니다. 적극적인 변이와 연관된 mRNAs의 총 수영장은 관련 microarray 4 깊은 시퀀싱 분석을 통해 결정 5하실 수 있습니다. 단백질 협회는 또한 시약 6 연결하는 십자가의 적절한 이외의 안정 수 있습니다. 포유 동물 세포에서 Polyribosome 분석도 안정 polysom​​es를 얻을하는 데 필요한 조건을 확립에 명백한 문제의 관점에서 서로 다른 프로토콜로 언급할 가치가있다. 문헌에 보고된 버퍼 시스템은, 7-10 널리 변동 때문에 용해 버퍼의 셀 타입 특정 최적화에 대한 요구 사항이있을 수도 있고, 그것은 버퍼 시스템 작업에 예민한 관심을만큼 중요하지 않습니다 것을 동등하게 그럴듯 신선한 lysates은 낮은 온도에서 보관. 우리의 지식 포유 동물 polysom​​e 형성에 영향을 미치는 매개 변수의 체계적인 평가는보고되지 않았습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

단백질 합성은 여러 수준에서 규제되는 복잡한 세포 과정이다. 예를 들어, 글로벌 번역 같은 아미노산 기아 또는 성장 인자의 탈퇴로 세포 스트레스 다양한 의해 개시 단계 또는 신장 단계에서 저해하실 수 있습니다. 또는, 개별 mRNAs의 번역은 mRNA의 현지화 또는 동족 microRNAs의 존재에 의해 조정할 수 있습니다. 단백질 합성의 연구 자주 번역 규칙 또는 단백질 합성에서 결함의 메카니즘에 대해 설명해 주실 polyribosome 분석을 활용합니다. 이 분석에서 mRNA / ribosome 단지는 진핵 세포에서 격리됩니다. 자당 밀도 기울기는 단일 ribosome 또는 monosome에 바운드 mRNAs에서 polyribosomes으로 알려진 여러 변이에 바운드 mRNAs을 분리합니다. 그라디언트의 분류는 고립과 다른 ribosomal 인구와 관련 mRNAs이나 단백질의 부량 수 있습니다. 정의된 조건 monosomes에 polyribosomes의 비율의 차이는 번역 개시 또는 신장 / 종료 중 하나에 결함을 나타내는 수 있습니다. polyribosome 분수에있는 mRNAs의 시험 개별 mRNAs의 일대는 실험 조건 변화를 번역되고 있는지 여부를 확인할 수 있습니다. 또한, ribosome 조립도 기울기로 구분하는 크고 작은 ribosomal subunit의 봉우리 분석하여 모니터링할 수 있습니다. 이 비디오에서는, 우리는 효모 세포, 자당 기울기에 의해 추출물의 분리 및 결과의 해석에서 원유 ribosomal 추출물의 준비를위한 방법을 제시한다. 이 절차는 포유 동물 세포에 쉽게 적용할 수 있습니다.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

이 문서는 진핵 세포의 변이를 번역의 추출을위한 프로토콜을 설명합니다. 일단 추출된, 리보솜은 다른 ribosomal 인구가 분석 수 있도록 자당 기울기 분별 (분리)에 의해 monosomes 및 polyribosomes로 구분됩니다. 따라서이 방법은 번역의 규정을 조사를위한 황금 표준입니다.

진핵세포 Polyribosome 프로필 분석

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.4K 조회수.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. 이 문서는 진핵 세포의 변이를 번역의 추출을위한 프로토콜을 설명합니다. 일단 추출된, 리보솜은 다른 ribosomal 인구가 분석 수 있도록 자당 기울기 분별 (분리)에 의해 monosomes 및 polyribosomes로 구분됩니다. 따라서이 방법은 번역의 규정을 조사를위한 황금 표준입니다.

비디오: Eukaryotic Polyribosome Profile Analysis

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

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

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

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

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

연구

생물학

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

C2C12 Myoblast 세포를 사용하여 Bioenergetic 프로필 실험

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

도구로 상호 빛과 전자 현미경 (클레멘 타인)는 Microinjected 분자와 진핵 세포 하위 세포 목표를 시각화하는 방법


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

탄소 고정 가능성 화학 층상 남극 호수 및 평가에서 미생물 진핵 농축 문화의 확립

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Zebrafish 태아의 대사 프로필 분석

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

대사 프로파일의 실시간 분석<em&gt; 전의 VIVO</em&gt; 마우스 장내 크립트 Organoid 문화

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

진핵 세포에서 염색체 외 원형 DNA의 게놈 차원의 정화

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Macroinvertebrates 사용 하 여 그룹별 수생 rDNA 뇌관의 유전 직감 콘텐츠 분석을 위한 실험실 프로토콜

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

스캐닝 전자 현미경 (SEM)에서 형태소 분석에 대 한 화학 건조를 사용 하 여 간결한 진 핵 유기 체의 준비

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

포스트모템 컴퓨팅 단층 촬영의 이미지 렌더링 기술: 좌초된 세타시안의 생물학적 건강과 프로필 평가

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...