이 작품은 국립 과학 재단 (부여 안돼. DBI 0,519,984)과 국립 보건원 (부여 안돼. 5R01GM078279)에 의해 지원되었다 ...

식물 1) 준비 <ol><li> Protoplasts는 효소를 소화 세포 벽의 오른쪽 혼합이 1을 사용된다는 가정하에 다양한 식물 종 및 조직에서 얻을 수 있습니다. 본격적인 실험이 수행되기 전에, 소재의 작은 규모의 소화는 조직의 protoplasting 효율성, 효소 등을 평가하고 셀 정렬에 대한 %로 긍정적인 세포를 추정하기 위해 좋습니다. 여기, 셀 타입 - 구체적으로 표현하는 녹색 형광 단백질 (GFP)을 사용하는 Arabidopsis thaliana의 모종의 뿌리에서 파생된 protoplasts. endodermis와 정지 센터는 P SCR로 표시됩니다 : GFP와 P WOX5에 의해 정지 센터 : : GFP 2,3 (그림 1). </li><li> 뿌리 (0.22 % W / V Murashige와 Skoog 기초 매체 [시그마], 1 %의 성장 매체로 통해 성장 수, 나일론 필터 (NITEX 250 μm의 메쉬)에, 모종은 (그림 2A 시그마) phytatrays에서 hydroponically 재배 W / V 자당, 0.05 % W / V MES [2 - (N - morpholino) ethanesulfonic의 산] 코와 산도 5.7). 또한, 식물이 수직으로 위치 1 % 한천 플레이트에 나일론 필터 (100 μm의 메쉬) (그림 2B) 위에 성장하실 수 있습니다. </li><li> 원하는 경우 뿌리의 수확뿐만 아니라 에이즈 상기 필터의 사용은 또한, 묘목 쉽게 추가 치료를 용이하게합니다. 필터는 묘목이 관심의 촉매로 보충하거나 새로운 phytatrays 한천 플레이트에 EN 메스를 전송할 수 있습니다. 예를 들어, phytatray 설정은 최대 Arabidopsis 모종의 질소 치료 4 transcriptional 응답 세포 유형의 특정 분석에 사용되었습니다. </li><li> 미세하여 형광 마커가 제대로 (자신이 치료에 의해 영향을받을 수있는 세포 유형 특정 마커로 치료 옵션을 사용하여 특히) 표시되었는지 확인하십시오. 이 경우에는 모종은 형광 현미경 (; 그림 1 니콘)에서 검사를하고 있습니다. 셀 유형 - 특정 형광 마커 라인이 정확하게 어떤 세포 유형이 표시됩니다 확인하고 표현의 다양성을 확인할 수 공촛점 현미경으로 처음 특성화되어야합니다. </li></ol> protoplasting 솔루션의 2) 준비 <ol><li> 1.25 % W / V Cellulase (Yakult), 0.3 % W / V Macerozyme (Yakult), 0.4 M D - mannitol, 20 MM MES와 demineralized 물 20 MM의 KCl을 (1 M 주식에서) 분해 및 5.7으로 산도를 조정 1 M 트리스 / HCL pH를 7.5로. 이 솔루션은 약간 흐린 것입니다. </li><li> 55 솔루션을 열 ° C 10 분 동안 (솔루션은 분명 바뀔 것이다)와 0.1 %를 추가하기 전에 실온까지 식지 W / V BSA (소 혈청 알부민), 10 MM CaCl 2, 5 MM β - 메르 캅 토 에탄올 . </li></ol> 3) 착취 및 식물 재료의 protoplasting <ol><li> 뿌리는 메스와 나일론 메쉬 그들을 근근이 살아가고하여 수확과 protoplasting 솔루션을 포함하고있는 플라스크에 입금됩니다. 일반적으로, protoplasting 솔루션의 10 ML은 1500 세들링 뿌리마다 사용됩니다. </li><li> 한 시간 만이라도 실온에서 부드럽게 flasks (75 RPM)를 흔들어. 더 이상 부화 시간은 원형질의 수율을 증가시킬 수 있습니다뿐만 아니라 유전자 발현에 대한 자체 protoplasting의 효과에 추가됩니다. </li><li> 40 μm의 셀 스트레이너 (BD 팔콘)로 원형질 솔루션을 필터링하고 원뿔 15 ML 튜브 (BD 팔콘)을 통해 솔루션을 나눕니다. </li><li> 이 원심 분리 속도가 사용된 protoplasts 종류의, 그들의 취약성과 효소 치료 기간 동안 생산 세포 파편의 크기에 따라 달라집니다 500 G. 주에서 10 분 동안 스윙 - 버킷 원심 분리기에 튜브를 스핀. </li><li> 뜨는의 대부분을 제거 나머지 솔루션 protoplasts을 resuspend하고 (그림 3) 미세 그들을 검사합니다. </li><li> protoplasts의 수와 밀도를 추정하는 hemacytometer을 사용합니다. 세포 밀도는 샘플 주입 속도, 외과에서 세포를 정렬하는 데 필요한 초 속도 당 행사와 따라서 전체 시간을 (또한 섹션 4.3 참조) 결정됩니다. </li><li> 3.7)은 외과로 바로 이동하거나없이 W5 (154 MM NaCl, 125 MM CaCl2, 5 MM KCl, 5 MM MES, 코와 5.7으로 산도를 조정) 또는 protoplasting 솔루션으로 부화 솔루션에 protoplasts 씻고 resuspend거나 효소를 추가했습니다. transcriptional 변경보고 특히 때에는 protoplasts 그것은 따라서 protoplasting 솔루션 protoplasts를 유지하고 진행하는 것이 좋습니다 인치 보관하는 버퍼를 변경으로 유전자 발현에 영향을 미칠 수 조건에 샘플의 노출을 최소화하기 위해 중요 가능한 한 빨리 외과합니다. </li></ol> protoplasts 4) 형광 활성 셀 정렬 <ol><li> 전원을 켜고 세포 분류기를 준비합니다. 여기 FACSAria (BD)가 사용됩니다. </li><li> 100 &amp; M과 흐름을 설정U; m 노즐과 20 PSI의 드레스 압력. </li><li> 세포 밀도와 샘플 사출 속도는 최상의 수율이나 빠른 속도를 원하는 달성 여부에 따라 특정 실험을 조정할 수 있습니다. 우리는 천만 셀 / ML 최대 밀도와 함께 성공적으로 정렬합니다. </li><li> protoplasts의 침강을 방지하기 위해 외과에 샘플 교반 옵션을 사용합니다. 1 : 외과의 막히는 것은 문제가있다면, 세 가지 문제 해결 단계가 있습니다. 샘플 온라인 backflush를 수행합니다. 2. 밀도를 줄이기 위해 귀하의 원형질 현탁액을 희석. 3. 원심 분리 및 resuspension 후 여과 단계 (3.3)를 반복하여 원형질의 솔루션을 정리. </li><li> 488 NM 레이저에 의해 여기 이후 붉은 스펙트럼 autofluorescence (RSA)에 GFP 및 20분의 610 nm의에 대한 30분의 530 nm의에서 앞으로 분산형 (FSC), 사이드 분산형 (SSC)와 방출을 측정하는 장치를 준비합니다. 이들은 본질적으로 GFP 양성 protoplasts을 분리하는 데 사용되는 유일한 매개 변수입니다. 여기 전압 설정은 다음과 같이되었습니다 : FSC - 60V, SSC 250V, 350V GFP 및 RSA 335V. 최적의 전압 설정은 모든 외과에 대해 서로 다른 것입니다 심지어 세포 분류기의 수명에 걸쳐 조정해야됩니다. </li><li> 앞으로 산란 대 사이드 분산을위한 dotplot를 설정하여 시작합니다. 측정된 이벤트 줄거리 중심으로되도록 전압 설정을 적용합니다. </li><li> 다음, 녹색 적색 형광 신호 대의 점 음모를 만들 수 있습니다. 야생 형 (비 GFP) 원형질의 정지를보고 때 측정 사건 줄거리의 중심 대각선 인구를 얻을 수 있도록 전압 설정을 적용합니다. GFP 표식 라인에서 파생된 원형질 서스펜션은 거친 타입 샘플에서 본 적이 녹색 형광 사건의 명확한 인구를 생성합니다. </li><li> GFP와 RSA 사이의 스펙트럼 중복에 대한 조정 보상 제약을 설정합니다. 적절한 보상 제약 설정은 비 GFP의 protoplasts와 부스러기에서 GFP 양성 protoplasts보다 분리 수 있습니다. RSA, 마이너스 17.91 %의 GFP : 여기에 사용되는 제약 조건은 다음과 같다. </li><li> 비 GFP의 protoplasts의 부정적인 컨트롤 게이트 경계 (그림 4) 정의에 투입 사용해야 GFP - 긍정적인 이벤트를 식별하기 위해 게이트를 설정합니다. </li><li> 분석에서 작은 파편를 떠나하기 위해 앞으로 흩어 커토프를 구현합니다. 컷오프의 위치를​​ 결정하는 데 도움이되도록 FSC 대 SSC의 음모에 GFP - 긍정적인 이벤트를 시각화. 여기서 단절는 5000으로 설정했습니다. 외과 조각이 높은 수준 정렬 행사와 샘플로 파편 계산됩니다이 예상보다 다른 %의 GFP 긍정적인 이벤트를 할 수 있습니다. 이것은 문제가 아니다. 그러나, 예제에서 더 많은 파편은 더 이상 정렬이됩니다. </li><li> 실험 및 분석에 대한 셀 타입의 풍부한에 따라 정렬 세포의 최적의 수율이나 최적의 순도에 대한 어느 외과 정밀 모드를 설정합니다. </li><li> RNA 추출을위한, RNA 추출 버퍼의 적절한 양의 컬렉션 튜브 (1.5 ML microfuge 튜브) 준비합니다. 이러한 설치로는, 20,000 정렬 이벤트 추출 버퍼 350 μl (RNeasy 마이크로 키트, QIAGEN)에 정렬되었습니다 약 100 μl의 총 볼륨을 얻을 것입니다. 정렬 완료 후 세포 현탁액은 상단에있는 수영장 수있는 샘플을 섞는다. </li><li> 샘플을 저장하거나 추출 RNA로 바로 진행합니다. 성공 microarray 분석은 같은 작은 500 정렬 행사에서 추출한 RNA로 preformed되었습니다. 여기, 우리는 RNeasy 마이크로 추출 키트 (QIAGEN) WT - 오베이션 피코 RNA의 증폭 시스템 및 FL - 오베이션 cDNA 비오틴 모듈 V2 (NuGEN)를 사용합니다. </li></ol> 대표 결과 약 1500 한 주 된 P SCR 중 하나 phytatray가 : GFP의 모종은 60,000 protoplasts (같은 hemacytometer에 의해 측정)에 대한 굴복. 65,000 외과 - 처리 이벤트의 2.6 %는 GFP - 긍정적인 것으로 정의되었고 (그림 4B) 정렬되었습니다. 약 1,500 4 일 된 P WOX5의 여덟 접시 : GFP의 모종 각 (12,000 총) 30,000,000 protoplasts (같은 hemacytometer에 의해 측정)에 대한 굴복. 16,000,000 외과 - 처리 이벤트의 0.063 %의 GFP - 긍정적인 것으로 정의되었고 (그림 4C) 정렬되었습니다. 10,000 정렬 이벤트는 일반적으로 RNA 추출을 위해 사용되며 20 일부터 140 NG 총 RNA (그림 5)을 얻을 수 있습니다. <img src="/files/ftp_upload/1673/1673fig1.jpg" alt="그림 1" /> 그림 1. Arabidopsis 루트에 셀 유형 특정 GFP 표지 라인. 형광 현미경 이미지는 미분 간섭 대비 (DIC) 및 Metamorph 소프트웨어 (분자 ​​장치)에서 실행 이클립스 90i 현미경 (니콘)에 GFP 필터로 찍은되었습니다. DIC와 GFP 이미지 시각화 목적으로 겹쳐 있었다. 이 영상 실험에 사용되는 두 개의 마커 라인이 표시되며A) P SCR : GFP와 B) P WOX5 : GFP. <img src="/files/ftp_upload/1673/1673fig2.jpg" alt="그림 2" /> 그림 2. 식물 성장 조건을. 모종은 phytatrays (A) 또는 수직 위치를 한천 플레이트 (B)에 hydroponically 환경 컨트롤러에서 성장했다. <img src="/files/ftp_upload/1673/1673fig3.jpg" alt="그림 3" /> 그림 3. 플랜트 protoplasts는 GFP를 표현. 형광 현미경 이미지가 차등 간섭 대비 (DIC) 및 Metamorph 소프트웨어 (분자 ​​장치)에서 실행 이클립스 90i 현미경 (니콘)에 GFP 필터로 찍은되었습니다. DIC와 GFP 이미지 시각화 목적으로 겹쳐 있었다. 화살표는 버스트 세포, 세포 파편 및 GFP 양성 원형질을 나타냅니다. 이 흰색 라인 사이의 거리는 50 μm의 수 있습니다. <img src="/files/ftp_upload/1673/1673fig4.jpg" alt="그림 4" /> 그림 4. GFP 양성 protoplasts의 형광 활성 세포 분류 야생 유형 (A) P SCR에서 파생 Protoplasts이 :. : GFP (B) 또는 P WOX5 : GFP (C) 마커 라인이 FACSAria (BD)를 분석하고 정렬되었습니다 정의 게이트를 사용하여 대 레드 (20분의 610 NM; Y 축) 형광, 녹색 (X 축 30분의 530 NM)의 dotplot에. 100,000 이벤트가 각각의 음모에 제공됩니다. GFP의 분류 게이트 안에 떨어지는 이벤트가 녹색으로 강조 표시됩니다. <img src="/files/ftp_upload/1673/1673fig5.jpg" alt="그림 5" /> 그림 5. 10.000 정렬 세포에서 RNA extractions 대표. 세포 RNA 추출 버퍼 (QIAGEN)에 직접 정렬되었습니다는 RNA는 정화와 2100 Bioanalyzer (애질런트)에 집중, 순도와 무결성을 확인했습니다. 두 마커 라인이 표시됩니다 세는 복제합니다.

<ol>
	<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts.">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127, 1466-1475 (2001).</li><li>Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+SCARECROW+function+reveals+a+radial+patterning+mechanism+common+to+root+and+shoot.">Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.</a> Development. 127, 595-603 (2000).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+nitrogen+responses+mediate+developmental+plasticity.">Cell-specific nitrogen responses mediate developmental plasticity.</a> Proc Natl Acad Sci U S A. 105, 803-808 (2008).</li><li>Bargmann, B. O. R., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+fluorescent+selection+permits+precise,+rapid,+and+in-depth+overexpression+analysis+in+plant+protoplasts.">Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.</a> Plant Physiol. 149, 1231-1239 (2009).</li><li>Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., Ljung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Auxin+Gradient+and+Maximum+in+the+Arabidopsis+Root+Apex+Shown+by+High-Resolution+Cell-Specific+Analysis+of+IAA+Distribution+and+Synthesis.">An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis.</a> Plant Cell. 21, 1659-1668 (2009).</li></ol>

Protoplasts는 원칙적으로 유리한 조건을 최적화하는 것은 크게 RNA의 품질과 수량을 향상시키는, 식물 조직의 다양한에서 파생된 수 있습니다. protoplasting 솔루션 및 사용 선택 부화 버퍼 모두이 부분에 영향을 미칠 것이다. 여러 가지 형광 단백질을 사용 외과의 기능에 따라 사용할 수 있습니다, 예를 들어 GFP, RFP, YFP, CFP하거나 많은 변종 및 파생 상품. 마커의 표현이 여기...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>250 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-250/50</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-100/47</td>
<td>&nbsp;</td>
<tr>
<td>Square petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-10k</td>
<td>&nbsp;</td>
<tr>
<td>Phytatrays</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1552</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog Basal Medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S5-3</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2933</td>
<td>&nbsp;</td>
<tr>
<td>KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1767</td>
<td>10 M stock</td>
</tr>
<tr>
<td>Eclipse 90i microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8041</td>
<td>1 M stock</td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3912</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>CALBIOCHEM</td>
<td>444203</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2536</td>
<td> 1 M stock</td>
</tr>
<tr>
<td>orbital shaker</td>
<td>&nbsp;</td>
<td>LAB-LINE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>conical 15 ml tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352196</td>
<td>&nbsp;</td>
<tr>
<td>table centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>Legend RT</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>FACSAria</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20170-38</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy micro kit</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>74004</td>
<td>&nbsp;</td>
<tr>
<td>WT-Ovation Pico RNA Amplification System</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>3300_12</td>
<td>&nbsp;</td>
<tr>
<td>FL-Ovation cDNA Biotin Module V2</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>4200_12</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence activated cell sorting of plant protoplasts

높은 해상도, 유전자 발현의 세포 유형의 특정 분석은 크게 발달 규제 및 모든 다세포 생물 환경 자극에 반응의 이해를 향상시킵니다. 원위치 하이브리드화와 리포터 유전자의 시각화에서 양적이를 데 사용 제한 범위 수 있지만 높은 해상도 RT - PCR 또는 특정 세포 유형에서 높은 처리량 transcriptome - 다양한 분석 RNA의 격리가 필요한 것입니다. 특정 세포 유형과 후속 형광 활성 세포 정렬 (외과)에 형광 단백질 마커를 표현하는 조직의 세포 분리는 가능한 RNA 추출, cDNA 합성 / 증폭 및 microarray 분석을위한 자료의 충분한 양을를 수집합니다. 셀 유형 - 특정 형광 기자 라인의 광범위한 집합은 식물 연구 커뮤니티에 사용할 수 있습니다. 이 경우, Arabidopsis thaliana 루트 두 마커 라인이 사용됩니다 : P SCR : GFP (endodermis 및 정지 센터)와 P WOX5 : GFP를 (정지 센터). 모종 많은 (수천) hydroponically 또는 한천 플레이트에 성장하고 추가 분석을 위해 충분한 루트 자료를 얻기 위해 수확하고 있습니다. 식물의 세포 분열은 세포 벽 소화 효소에 의해 이루어진다. 이 절차는 높은 osmolarity - 유도 plasmolysis 및 상용 cellulases을 사용하게, pectinases 및 hemicellulases이 솔루션에 protoplasts을 해제합니다. GFP 양성 세포 외과는 488 nm의 레이저에 의해 흥분 protoplasts의 붉은 방출 스펙트럼 대 녹색의 시각화을 사용합니다. GFP 양성 protoplasts은 빨간색으로 방출하는 녹색들의 증가 비율로 구분할 수 있습니다. Protoplasts는 일반적으로 RNA 추출 버퍼에 직접 정렬하고 나중에 추가 처리를 위해 저장됩니다. 이 기술은 간단하고 실용적인 것으로 드러났습니다. 또한, 그것은 심지어 매우 부족한 세포 유형 (예 : 정지 중심 세포)에 대한, 그것이 transcriptome 분석을 위해 세포의 충분한 숫자를 분리하는 어려움없이 사용할 수 표시됩니다. 마지막으로, Arabidopsis 모종의 성장 설정은 그 (셀 타입 - 특정 biotic 또는 비생물적 스트레스 응답의 분석 예) 셀 정렬하기 전에 식물의 단순한 치료를 가능하게 증명합니다. 잠재적인 보조는 식물 protoplasts의 외과에 대해 설명합니다.

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting of Plant Protoplasts at JoVE.com

Fluorescence Activated Cell Sorting of Plant Protoplasts

식물의 특정 세포 유형을 분리하는 방법을 시연합니다. 이 기법은 특정 세포 유형, 세포 분리 및 형광 활성 세포에 형광 단백질 정렬을 표현 형질 마커 라인을 사용합니다. 또한, 성장의 설정은 치료를 용이하게되는 여기 설립<em&gt; Arabidopsis thaliana</em셀 정렬하기 전에&gt; 모종.

식물 Protoplasts의 형광 활성 세포 정렬

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

fluorescence-activated-cell-sorting-of-plant-protoplasts

Research

JoVE Journal

Biology

24.7K 조회수.  New York University. 식물의 특정 세포 유형을 분리하는 방법을 시연합니다. 이 기법은 특정 세포 유형, 세포 분리 및 형광 활성 세포에 형광 단백질 정렬을 표현 형질 마커 라인을 사용합니다. 또한, 성장의 설정은 치료를 용이하게되는 여기 설립 Arabidopsis thaliana 모종.

비디오: Fluorescence Activated Cell Sorting of Plant Protoplasts

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

자기 활성 셀 정렬 (LIMACS)를 사용하여 지질 소포 - 중재 동질 크로마 토그래피 : 단백질 지질 상호 작용을 분석하는 소설 방법

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

연구

생물학

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxyuridine (BrdU) 레이블과 녹아 유기 문화 독립적인 확인을위한 후속 형광 활성 세포 정렬 탄소 굴욕 Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

의 세포 구체적인 분석<em&gt; Arabidopsis</em&gt; 형광 활성화 셀 정렬을 사용하여 잎

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

셀 추적 염료를 사용하여 전지 부문 모니터링에 최적화 된 착색 및 확산 모델링 방법

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

형광 활성 세포 정렬 (FACS)에 의해 신선한 조직으로부터 정상 및 암 관련 섬유 아세포의 분리

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

담배 원형질체 및 잎에 이분자 형광 보완에 의해 시각 단백질 - 단백질 상호 작용

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

삼투 물 투수 계수 (P 측정<sub&gt; F</sub&gt;) 구형 세포 : 예를 들어 식물 절연 원형질체

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

발광 시계 기자와 애기 장대 원형질체 트랜의 일주기 표현형의 신속한 분석

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...