Anyaporn Sawasdichai 및 Nazefah 압둘 하미드는 박사에 대한 각각 로얄 태국 정부와 말레이시아 정부에 감사 장학금. 이 작품은 PSJ와 KG에게 수여 웰컴 트러스트 프로젝트 부여하여 자금을했습니다. 우리는 또한 브리스톨 울프슨의 Bioimaging 시설의 대학에 감사하고 있습니다.

분류에 대한 I. 준비 이 섹션은 폴리 - L - 라이신 코팅 현미경 coverslips 및 분류하기 전에 세포의 첨부 파일의 준비를 설명합니다. 필요한 경우 전지 transiently 첨부 앞 또는 뒤에 하나 단백질 발현 벡터와 함께 transfected 수 있습니다. 폴리 - L - 라이신 코팅 coverslips의 A. 준비 <ol><li> 증류수에 1mg/ml 폴리 - L - 라이신의 솔루션을 준비합니다. </li><li> 22 락을 플랫폼에서 최소한 1 시간 동안 솔루션에서 그들을 잠복기로 폴리 - L - 라이신과 겉옷 깨끗한 coverslips ° C. </li><li> 두 멸균 증류수로 코팅 coverslips를 씻고 96 % 에탄올과 함께 하나의 세척과 함께하십시오. </li><li> 에어 필터 종이에 코팅 coverslips을 건조하고 나중에 사용할 수 드라이 컨테이너 (일단 드라이들은 스택 가능)에 보관하십시오. </li></ol> coverslips에 B. 첨​​부 세포 비 점착 성의 세포 (여기서 우리는 K562 세포를 사용) <ol><li> 최대 직면한 코팅 표면을 가진 6 잘 플레이트에 잘에서 폴리 - L - 라이신 코팅 coverslip를 놓습니다. </li><li> 7x10 5 세포의 밀도에 씨앗 K562 세포 / 잘 Dulbecco의 수정 이글스 중간 (DMEM) 10 % 태아 소 혈청 (FBS) 및 PS (페니실린 100units/ml, 스트렙토 마이신의 100mg/ml)와 보충 인치 K562 세포의 경우에는 과도 transfection은 세포를 시딩 전에 electroporation을 (250V / 975μF에서 매체의 200μl에 1x10 7 전지 0.4cm electroporation의 cuvettes)를 사용하여 수행할 수 있습니다. </li><li> 37 ° C 5 % CO 2에서 24 시간 동안 세포를 품어. </li><li> 매체를 붓고 얼음 차가운 인산과 함께 두 번 세포 세차 호수 (PBS)를 버퍼. </li><li> subcellular 분획화 프로토콜하십시​​오. </li></ol> 자기편 세포 (여기서 우리가 만들었기에 - 7 세포를 사용) <ol><li> 6 잘 판에있는 우물에 uncoated coverslip를 놓습니다. </li><li> PBS로 두 번 씻어 37 ° C.에 prewarmed </li><li> 37 PBS의 트립신 (0.03 % EDTA (에틸렌 다이아 민 테트라 초산), 0.25 %의 트립신)를 소화하여 세포를 분리 ° C 2~5분하십시오. 그것은 주로 개인 떠있는 세포가 존재로 전지를 오버 소화하고 즉시 소화를 중지하지 않는 것이 중요합니다. </li><li> / 잘 10% FBS와 PSG (페니실린 100 단위 / ML, 스트렙토 마이신 100ug/ml 및 L - 글루타민 292ug/ml)와 보충 DMEM에 6 잘 판에 3x10 5 세포의 밀도에 세포를 시딩하여 소화를 중지합니다 정상 coverslips에. 자기편 세포는 일반적으로 필요한 경우에는, 폴리 - L - 라이신 코팅 coverslips 사용할 수있는 정상적인 현미경 coverslips에 잘 부착합니다. </li><li> 37 ° C와 5% CO 2에서 24 시간 동안 세포를 품어. 왜냐하면 - 7 세포의 경우에는 과도 transfection은 Fugene 6 (로체) 필요한 경우 37에서 추가로 24 시간 동안 복구 왼쪽으로 세포를 사용하여 수행할 수 있습니다 ° C와 5 % CO 2. </li><li> 매체를 붓고 얼음 차가운 PBS로 두 번 세포를 씻으십시오. </li><li> subcellular 분획화 프로토콜하십시​​오. </li></ol> II. Subcellular 분별 (분리) 현장 분획에서는 그림 1에 표시된 설계도에 따라 수행되고있는 방법을 기반으로 함께 이전 5 설명한 변경 사항은 이전에 7 아래의 자세한 프로토콜 설명했다. 이것은 액체를 제거하기 전에 coverslip을 제거 각 분류 단계 다음과 같은 새로운 6 - 잘 판에 coverslips을 전송하는 것이 좋습니다. 적당히 coverslips이 영상에 필요한 있지만, 추가 coverslips이가 병렬로 진행하는 경우 서부는 모래 바닥에 대한 전체 세포 추출물과 핵 매트릭스 분수를 생성해야합니다. <ol><li> 준비하고 cytoskeleton 버퍼 (CSK) 필터 : 10mM 파이프, 300mM 자당, 100mM NaCl, 3mM MgCl 2, 1mM EGTA합니다. CSK 버퍼는 신선한 분별화 당일 준비되어야합니다. </li><li> 필요한 경우 하나 coverslip에서 세포가 부드럽게 직접 coverslip에 200ul의 TES 버퍼 (1 % SDS, 2mM EDTA (에틸렌 다이아 민 테트라 초산), 20mM 트리스 - HCL 산도 7.4)을 배치하고 1 ~ 2 분 동안 잠복기에 의해 모래 바닥 서부에 대한 전체 세포 추출물을 준비하는 데 사용할 수 있습니다 22 ° C. 전체 세포 추출물은 다음 1M의 250ul (NH 4) 2 SO 4 추가하고 다른 서양의 샘플이 준비되기 전까지는 얼음 잠복기에 의해 제거되고 단백질은 시켰던 수 있습니다. </li><li> 22 ° C 기울이기로 6 잘 플레이트 두세 시간에 두 번 1ml 얼음 차가운 PBS로 남아 coverslips 씻으십시오. </li><li> 전체 세포와 신선한 6 잘 판에 전송을 대표하는 coverslip을 제거합니다. 세포 고정과 단백질 형광 프로토콜과 함께하십시오. </li><li> 부드럽게 남아있는 우물에서 PBS를 제거합니다. </li><li> 부드럽게 200ul CSK 버퍼를 배치하여 세포질과 핵 단백질 개최 느슨하게 압축을 풉니다 + 0.1 % (V / V) 트리톤 직접 각 나머지 coverslip에 X - 100 잠복기에1 분 얼음. </li><li> 세포질과 느슨하게 개최 핵 추출물을 제거하고이 단계는 2 단계에 설명된대로 침전물. </li><li> 22 회 1ml 얼음 차가운 PBS로 c​​overslips을 씻어 ° C를 기울이기 6 잘 판에 의해. </li><li> 밀접하게 개최 핵 물질을 대표하는 coverslip을 제거하고 세포 고정과 단백질 형광 프로토콜과 함께하십시오. </li><li> 부드럽게 남아있는 우물에서 PBS를 제거한 후 부드럽게 + 0.5 % (V / V) 트리톤 X - 100에 직접 각 나머지 coverslip에 200ul CSK 버퍼를 넣어 20 분 동안 얼음 잠복기에 의해 단단히 개최 단백질을 추출합니다. </li><li> 단단히 개최 추출물을 제거하고 2 단계에서 설명한대로 침전물. </li><li> 22 회 1ml 얼음 차가운 PBS로 c​​overslips을 씻어 ° C를 기울이기 6 잘 판에 의해. </li><li> 염색질 분획을 대표하는 coverslip을 제거하고 세포 고정과 단백질 형광 프로토콜하십시​​오. </li><li> 부드럽게 다음 장소 200ul CSK 버퍼 + 100ug/ml DNase I 30 분 동안 나머지 coverslips과 부화에 37 ° C. 남아있는 우물에서 PBS를 제거 </li><li> 염색질 추출물을 제거하고 2 단계에서 설명한대로 침전물. </li><li> ° C 틸팅하여 6 잘 플레이트 22 회 얼음 차가운 PBS로 c​​overslips 씻으십시오. </li><li> 핵 매트릭스 분율을 나타내는 coverslip을 제거하고 세포 고정과 단백질 형광 프로토콜과 함께하십시오. </li><li> 필요한 경우 매트릭스 단백질은 2 단계에서 설명한대로 200ul의 TES 버퍼를 사용하여 모래 바닥 서부에 대한 추가 매트릭스 coverslip에서 제거할 수 있습니다. </li><li> 서양 얼룩에 대한 샘플은 4 벤치 최고 microcentrifuge에서 13,000 rpm으로 centrifuged되어야 ° C와 40ul SDS 로딩 버퍼 (62.5mM 트리스 - HCL 산도 6.8, 2퍼센트 SDS (W / V), 50 MM DTT에 resuspended 산탄 10 % 글리세롤, SDS - PAGE로 분석하기 전에 0.01 % bromophenol 블루 (W / V)). <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="그림 1" /> 그림 1. 현장 subcellular 분별 (분리)에의 개략도 표현. Cytoskeleton 버퍼 (CSK)는 10mM 파이프, 300mM 자당, 100mM NaCl, 3mM MgCl 2, 1mM EGTA으로 구성되어 있습니다. TES 버퍼가 1 % SDS, 2mM EDTA (에틸렌 다이아 민 테트라 초산), 20mM 트리스 - HCL pH를 7.4로 구성되어 있습니다. </li></ol> III. 세포 고정과 단백질 형광 <ol><li> 부드럽게 30 분 4 각 coverslip과 부화 ° C ~ 4 %의 포름 알데히드 / PBS의 1ml를 추가합니다. </li><li> 각 coverslip에게 22 PBS의 1ml로 부드럽게 2-3 회 ° C. 와시 </li><li> 10 분 22 ° C에서 각 coverslip과 부화 0.1 % 트리톤 X-100/PBS의 400ul를 추가합니다. </li><li> 각 coverslip에게 22 PBS의 1ml로 부드럽게 2-3 회 ° C. 와시 </li><li> 잠재적인 배경 얼룩을 줄이기 위해 20 분 동안 22 ° C에서 3퍼센트 소 혈청 알부민 (BSA) / PBS와 부화의 400ul를 추가합니다. </li><li> 각 coverslip에게 22 PBS의 1ml로 부드럽게 2-3 회 ° C. 와시 </li><li> 직접 1 시간 22 ° C에서 coverslip과 부화에 PBS에서 기본 항체의 플레이스 100ul. 기본 및 보조 항체가 필요한 농도로 PBS에 희석한다. 이것은 사용되는 각각의 항체에 대한 최적화 할 수 있습니다. </li><li> 각 coverslip에게 22 PBS의 1ml로 부드럽게 2-3 회 ° C. 와시 </li><li> ° C 떨어진 1 시간 빛으로부터 22 PBS과 부화에 이차 항체의 플레이스 200ul. </li><li> 각 coverslip에게 22 PBS의 1ml로 부드럽게 2-3 회 ° C. 와시 </li><li> DAPI (4 &#39;, 6 diamidino - 2 - phenylindole 염산염)이있는 Vectashield 장착 매체를 사용하여 유리 현미경 슬라이드에 각각 coverslip을 (셀 아래) 마운트합니다. </li><li> 종이 타월을 사용하여 coverslip 주위에서 초과 장착 매체를 닦고 적어도 30 분 거리 빛으로부터 22 ° C에서 슬라이드를 품어. </li><li> 명확한 아크릴 매니큐어를 사용하여 유리 슬라이드에 coverslip을 수정. </li><li> 형광 현미경으로 시각화하기 전에 멀리 빛으로부터 슬라이드를 유지. </li></ol> IV. 대표 결과 <img src="/files/ftp_upload/1958/1958fig2.jpg" alt="그림 2" /> 그림 2. 비 transfected COS - 7 세포 Subcellular 분별 (분리). Fractionated 있으니까요 - 7 전지는 포름 알데히드와 고정 및 DAPI를 포함하는 설치 매체를 치료 다음 TRITC하는 복합 A / C 항체 α - Tubulin, α - 히스톤 H1 또는 α - Lamin를 사용 immunostained되었습니다 . 이미지 Leica 공촛점 현미경을 사용하여 10X 안구 렌즈와 63x 객관적인 렌즈로 인수했다. 세포 핵은 마커 항체 물들일 동일한 필드 셀이에 TRITC 필터 세트를 사용하여 시각 동안 DAPI 필터 세트를 사용하여 시각되었습니다. <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="그림 3" /> 그림 3. GFP - 6E2 융합 단백질은 핵 매트릭스와 연결되어 있습니다. 인간 papillomavirus (HPV) 유형 6 융합 녹색 형광 단백질 (GFP)로 구성된 융합 단백질을 표현 있으니까요 - 7 세포의 Subcellular 분별 (분리)E2 단백질 (GFP - 6E2). transfected 세포에 GFP - 6E2의 지방화는 GFP 형광을 통해 직접 시각 동안 세포 핵은 DAPI의 얼룩을 통해 시각되었습니다. Lamin A / C는 α - Lamin TRITC과 TRITC 필터 설정 복합 / C 항체를 사용하여 시각되었습니다. 병합 이미지는 GFP - 6E2 단백질 중 일부는 핵 매트릭스 (오른쪽 아래 패널)와 관련된 것을 밝히지. 그림 2에서 설명한대로 이미지를 취득했다.

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	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

관심 없음 충돌 선언하지 않습니다.

일반적인 문제 및 제안 : 전부 또는 세포의 대부분은 세척하는 동안 손실됩니다. 세척 단계를하는 동안 액체는 coverslip을 피하 6 잘 접시의 측면으로 천천히 추가해야합니다. 마찬가지로 액체는 신중하게 틸팅 판에 의해 제거되어야하며 천천히 초과 액체를 pipetting. 세포 접착은 폴리 - L - 라이신 코팅 coverslips을 사용하여 증가 수 있습니다. 게놈 DNA가 완전?...

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<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>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in situ subcellular fractionation of adherent and non-adherent mammalian cells

단백질 기능을 속속들이 단백질 현지화로 결합됩니다. 어떤 단백질은 특정 위치 또는 subcellular 구획로 제한하고 있지만, 많은 단백질이 밀접하게 특정 subcellular 도메인 또는 구조와 관련된 하위 인구를 무료로 교환 자유롭게 diffusing 인구로 존재한다. 원위치 subcellular 분별 (분리)에서의 시각화가 가능 단백질 compartmentalization 또한 단백질 구체적인 구조에 집중 하위 인구를 공개하실 수 있습니다. 예를 들어, 용해 세포질 단백질과 느슨하게 개최 핵 단백질의 제거는 염색질와 함께 전사 요소의 안정적인 연결을 확인할 수 있습니다. DNA의 소화 이후 어떤 경우에는 집합 핵 발판이나 핵 매트릭스를 칭했다는 단백질과 RNAS의 네트워크 연결을 확인할 수 있습니다. 여기 현미경 coverslips에 자기편 및 비 점착 성의 포유 동물 세포의 현장 분별화에서 중 필요한 단계를 설명합니다. 단백질 시각화는 특정 항체 또는 형광 융합 단백질과 형광 현미경을 사용하여 얻을 수 있습니다. 항체 및 / 또는 특정 구획이나 구조에 대한 마커 역할을 형광 염료는 단백질 지방화는 자세히 매핑 될 수 있습니다. 원위치 분별화에서도 서양 각 분획에있는 단백질의 양을 비교 모래 바닥과 함께하실 수 있습니다. 이 간단한 생화 학적 접근 방법은 별도로 들키지 않고 남아있는 것이 연결을 확인할 수 있습니다.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

현미경 coverslips에서 포유 동물 세포의 현장 subcellular 분별 (분리)에서 단백질 지방화의 시각이 있습니다.

자기편 및 비 점착 성의 포유 동물 세포의 현장 Subcellular 분별 (분리)에서

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

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Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

15.7K 조회수.  University of Bristol. 현미경 coverslips에서 포유 동물 세포의 현장 subcellular 분별 (분리)에서 단백질 지방화의 시각이 있습니다.

비디오: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

포유 동물 세포를 Trypsinizing 및 Subculturing

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

연구

생물학

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic 분석 : 자기편 셀

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

포유 동물 세포의 세포주기의 위치 분석

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

포유류의 세포에서 Endoplasmic 소포체 현지화 mRNAs의 시각화

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

게놈 전체 규모에 Polysom​​e 분별과 포유류 Translatomes 분석

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

포유 동물 세포에서 헴 합성 레벨 측정

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

이미징 로컬 CA<sup&gt; 2 +</sup&gt; 배양 된 포유 동물 세포에서 신호

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...