이 작품은 슬로베니아어 연구기구 (프로젝트 J2 - 9764 및 프로그램 P2 - 0249)에 의해 지원되었다. 이 비디오는 &quot;Electroporation 기반 기술 및 트리 트먼트&quot;류블랴나, 슬로베니아 대학 전기 공학부 주최 과학 워크샵 및 대학원 과정에 대한 보충 자료를 나타냅니다.

나도 셀 추적기 CMFDA과 CMRA으로 세포 중 <ol><li> 실험 마우스 흑색증 셀 라인 (B16 - F1)의 이전 준비 세포에서 수행되었다. 세포 10% 태아 소 혈청, 0.15 MG / ML L - 글루타민, 16 MG와 보충 DMEM 문화 매체 70~80%의 합류 (Dulbecco의 수정 이글의 중간)에 두 분리된 25cm 두 문화 flasks (TPP, ZDA)에 재배 / ML gentamicin (시그마 - 알드리치, 독일에서), 200 단위 / ML crystacillin (Pliva, 크로아티아), 그리고 37 5 % CO 2에서 incubated ° C. </li><li> 원래의 염료 50 μg에 10.76 μl 및 DMSO (시그마 - 알드리치, 독일) 9 μl를 (그린 CMFDA과 오렌지 CMRA 들어, 각각)을 추가하여 셀 추적기의 두 10 MM 주식 솔루션 (Invitrogen, 미국)를 준비 Invitrogen의 유리병. 주식 솔루션 ° C 몇 달 동안 4 냉장고에 저장할 수 있습니다. DMSO의 크리스탈 녹이신 때까지 실험을 시작하기 전에, 솔루션을 따뜻하게. </li><li> 중탄산염 무료 크렙스 - Hepes 버퍼 (130 MM NaCl, 4.7 MM KCl, 1.2 MM MgSO 4, 1.2 MM KH 2 PO 4, 11.7 MM D - 글루코오스, 1.3 MM CaCl 2, 10 MM HEPES, pH를 7.4) 준비합니다. 두 15 ML Eppendorf 튜브는 별도로 중탄산염 무료 크렙스 - Hepes 버퍼 3 ML 각 재고 솔루션 2.1 μl (10 MM CMFDA 또는 CMRA, 각각)을 섞는다. 이것은 약 7 μm의 CMFDA (또는 CMRA)가 포함된 &#39;로딩 솔루션 &quot;을 산출. </li><li> 중탄산염 무료 크렙스 - Hepes 버퍼와 두 세포를 씻어 후 flasks에 솔루션을 로딩 삽입합니다. 37 5% CO 2에서 30 분 동안 전지를 품어 ° C. 이 첫 번째 배양 시약은 세포 점막을 통해 자유롭게 통과하는 동안, 일단 세포 내부의 시약은 휴대 impermeant 형광 반응 제품으로 변모하고 있습니다. </li><li> 37 첫번째 부화 후 씻어 5% CO 2에있는 다른 두 시간 동안 배지로 세포를 품어 ° C. </li><li> 두 flasks의 세포를 Trypsinize (CMFDA 및 CMRA와 함께로드)과 50 ML의 원심 분리기 튜브의 비율 1시 1분 (TPP, ZDA)에 함께 적색과 녹색의 세포를 섞는다. DMEM 매체로 희석하거나 원심 분리기와 농도가 5 × 10 6 세포 / ML에 세포 농도를 조정합니다. 24 multiwell 플레이트 (TPP, ZDA) 각 우물에 세포 현탁액의 20 μl 드롭 놓습니다. ° C 20 분 위해 약간 우물의 표면에 부착하고 세포 연락처를 설정할 수 있도록 37 5 % CO 2 세포를 품어. </li></ol> II. Electrofusion <ol><li> (10 MM KH2PO4, 10 MM K 2 HPO 4, 1mM MgCl 2, 75 MM의 자당) isoosomolar 칼륨 인산 버퍼를 준비 (10 MM KH2PO4, 10 MM K 2 HPO 4, 1mM MgCl 2, 250 MM의 자당)과 hypoosmolar 칼륨 인산염 버퍼 . </li><li> 잘 하단의 현미경 무대, 위치에 전지 전극으로 multiwell를 삽입하고 펄스 발생기에 연결합니다. </li><li> isoosomolar 인산 칼륨 완충액 1 ML있는 세포를 씻으십시오. 세포 팽창을 유도하기 위해 hypoosmolar 인산 칼륨 완충액 350 μl를 추가합니다. 버퍼 전극을 커버한다. </li><li> 전기 펄스를 적용하기 전에 2 분 동안 hypoosmolar 버퍼에 세포를 남겨주세요. 인테리어와 세포의 외부 사이의 삼투 불균형에 의한 세포에 물 분자의 시간 동안 유입 세포 볼륨의 증가의 원인이됩니다. 세포가 자신의 최대 볼륨을 가까이 때 전기 펄스는 규제 볼륨 감소 시작하기 전에 적용되어야합니다. </li><li> 최적의 electrofusion를 달성하고 세포 생존을 유지하기 위해 전기 펄스의 최적의 매개 변수를 사용해야합니다. 이들은 [1]를 사용하여 세포주에 따라 달라집니다. 이 실험에 8 직사각형 펄스 (1 Hz에서 100 μs 기간 각)의 기차는 electroporation 장치를 (우리의 경우 Cliniporator, IGEA, 이탈리아)를 사용하여 각 샘플에 적용됩니다. 펄스는 잘 컨트롤을 제외하고, 각 우물에 전극 사이에 약 1,200 V / cm의 전기장을 만들고, 0.8 mm의 직경과 그들 사이에 5mm 거리 두 개의 병렬 PT / IR 와이어 전극에 전달됩니다. </li><li> 펄스 전송 후 10 분 동안 그대로 세포를 남겨주세요. 형광 및 위상 대비 현미경을 통해 퓨전 수율을 결정합니다. </li></ol> III. 이미지 수집 및 융합 수율의 결정 <ol><li> 세포는 객관적 (x20) 및 냉각 CCD 카메라 (VisiCam 1280, Visitron, 독일)이 장착된 형광 현미경 (우리의 경우에는 자이스 혈구 AxioVert 200 자이스 혈구, 독일)를 사용하여 관찰하고 있습니다. 이미지 MetaMorph 7.1.1 (분자 디바이스, 미국)에 인수 있지만, 다른 유사한 수집 소프트웨어도 사용할 수 있습니다. CMFDA는 548 nm의에서 492 nm의에서 단색 (폴리 크롬 IV, Visitron, 독일)와 CMRA와 흥분이다. CMFDA 및 CMRA의 형광은 (D605/55m, C 두 방사 535 nm의 중심 필터, 한 (HQ535/30m, CMFDA)과 510 nm의에서 다른 중심을 사용하여 획득한 것입니다MRA, 채도, 미국) 모두. 이색성 거울 (Q515LP)의 사용은 채널 크로스 이야기를하지 못했습니다. </li><li> 각 잘 다섯 무작위로 선택된 분야 세 가지 이미지 (위상 콘트라스트, 빨간색과 초록색 형광) 취득. 각각의 이미지 플렛에서 3 채널 이미지를 만듭니다. 이러한 이미지의 fluorescing 세포질은 세포 점막과 함께 볼 수 있습니다. 융합 세포 따라서 쉽게 [그림 1]을 결정하실 수 있습니다. </li><li> 각 3 채널 이미지의 모든 세 가지 유형의 세포 (빨강, 녹색 및 dually 형광)을 계산합니다. 각 이미지의 모든 세포의 숫자 dually 형광 세포의 수를 나누어 dually 형광 세포의 비율을 결정합니다. 퓨전 수율은 융합 세포의 절반이 감지되지 않습니다 때문에이 곱한 dually 형광 세포의 비율로 정의 (때 같은 색상 퓨즈의 세포).입니다 </li></ol> 대표 결과 <img src="/files/ftp_upload/1991/1991fig1.jpg" alt="그림 1" /> 그림 1 electrofusion 후 B16F1 세포의 세 채널 현미경 이미지 :. 위상 콘트라스트, CMRA 형광 (548 nm의 여기에서)와 CMFDA 형광 (492 nm의 여기에서), 객관적인 확대 20x

<ol>
	<li>&#268;emazar, M., Jarm, T., Miklav&#269;i&#269;, D., Ma&#269;ek-Lebar, A., Ihan, A., Kopitar, N. A., Ser&#353;a, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+electric-field+intensity+on+electropermeabilization+and+electrosensitivity+of+various+tumor-cell+lines+in+vitro.">Effect of electric-field intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro.</a> Electro. Magnetobiol. 17, 263-272 (1998).</li><li>Trontelj, K., Reber&#353;ek, M., Kandu&#353;er, M., &#269;urin &#353;erbec, V., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+bulk+cell+electrofusion+in+vitro+for+production+of+human-mouse+heterohybridoma+cells.">Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells.</a> Bioelectrochemistry. 74, 124-129 (2008).</li><li>Rols, M. -. P., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+electrically+induced+permeabilization+and+fusion+of+Chinese+hamster+ovary+cells+by+osmotic+pressure.">Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure.</a> Biochemistry. 29, 4561-4567 (1990).</li><li>Neil, G., Zimmermann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrofusion.">Electrofusion.</a> Methods in Enzymology. 220, 174-196 .</li><li>Abidor, I. G., Li, L. -. H., Hui, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+cell+pellets:+II.+Osmotic+properties,+electroporation,+and+related+phenomena:+Membrane+interactions.">Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: Membrane interactions.</a> Biophysical journal. 67, 427-435 (1994).</li><li>Jaroszeski, M. J., Gilbert, R., Fallon, P. G., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+facilitated+cell-cell+electrofusion.">Mechanically facilitated cell-cell electrofusion.</a> Biophys J. 67 (4), 1574-1581 (1994).</li><li>Ramos, C., Bonefant, D., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+hybridization+by+electrofusion+on+filters.">Cell hybridization by electrofusion on filters.</a> Analytical biochemistry. 302, 213-219 (2002).</li><li>Cao, Y., Yang, J., Yin, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+high-throughput+cell+electrofusion+in+a+microelectrode-array+chip.">Study of high-throughput cell electrofusion in a microelectrode-array chip.</a> Microfluid Nanofluid. 5, 669-675 (2008).</li><li>U&#353;aj, M., Trontelj, M., Kandu&#353;er, M., Miklav&#269;i&#269;, 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+size+dynamics+and+viability+of+cells+exposed+to+hypotonic+treatment+and+electroporation+for+electrofusion+optimization.">Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization.</a> Radiol. Oncol. 43, 108-119 (2009).</li><li>Teissi&#233;, J., Ramos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+electric+field+pulse+induced+long-lived+permeabilization+and+fusogenicity+in+cell+membranes.">Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.</a> Biophys. J. 74, 1889-1898 (1998).</li></ol>

관심 없음 충돌 선언하지 않습니다.

비 - 구체적으로, 예를 들어, 외부 전기 분야로를 융합 세포 점막의 능력은 생물학 생명 공학, 의학 및 연구에 중요합니다. 이러한 특이 현상이 융합은 매우 가치있는 하이브리드 전지와 같은 단클론 항체와 같은 자사 제품의 생산을 가능하게하고, 퓨전 [2]의 근본적인 메커니즘에 대한 정보를 제공합니다. 제대로 세포의 다른 유형이 조정될 수 있습니다 이후 Electrofusion은 잠재적으로 매우 효과적인 방법입니다. 가까운 신체 접촉 세포가 자신의 fusogenic 상태로 가지고있을 때 Electrofusion는 고전압 전기 펄스에 의해 (융합하는 경향) 이루어진다. electrofusion의 효율 electrofusion 과정의 두 부분에 영향을 미칠 다양한 매개 변수에 따라 달라집니다. electrofusion 과정의 첫 부분은 세포 사이의 가까운 신체 접촉의 성과이며, 어떤은 다른 방법으로 얻을 수있다 [3-8]. 준수 방법은 (합류로 성장하는 세포) 세포를 효율적으로 사이에 큰 영역에서 자발적으로 설립된 세포 접촉에 의해 사용될 수 있지만, 그것은 많은 핵 매우 큰 융합 세포를 생산하고 있습니다. 생존 가능성이 더 높습니다 그리고 작은 세포가 세포 분열 따위에 의해 번식 (2-5 핵 포함), (그림 1)를 얻을 수있다 어디에 우리는 수정된 준수 방법을 사용하고 있습니다. 세포 사이의 연락도 실험 [9]에서 사용 삼투 치료로 인해 세포의 삼투 부기의 혜택을 누릴 수 있습니다. electrofusion 과정의 두 번째 부분은 세포 점막의 fusogenic 상태의 달성이다. Fusogenic 상태는 멤브레인의 electropermeabilized 상태 (세포가 아닌 구체적으로 정상적으로 그대로 막 통과할 수없는 분자로 permeabilized 아르)와 잘 연결합니다 및 전기 펄스 (진폭, 길이, 횟수 및 빈도와) 같은 매개 변수에 의해 적용됩니다 [10] . 최적의 electroporation을 위해 필요한 전기 매개 변수의 값은 [1]과 electrofusion가 다른 세포와 세포 사이의 차이가 크기와 자신의 생물 학적 특성에 따라 달라집니다. 전기 매개 변수 따라서 융합을 얻기 위해, 융합 파트너로 사용하는 다른 세포 라인에 대한 최적화해야합니다.

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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>CMRA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C34551</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>CMFDA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C7025</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>DMSO</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>DMEM </td>
<td>Reagent</td>
<td>Sigma-Aldrich </td>
<td>D5671</td>
<td>Dulbecco's modified Eagle's medium</td>
</tr>
<tr>
<td>Fetal calf serum</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>Reagent</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Hepes</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Merck </td>
<td>A124873 927</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>4248</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M-8266</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fluka</td>
<td>71382</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Merck</td>
<td>A154336 908</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M2643</td>
<td>&nbsp;</td>
<tr>
<td>D-glucose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C4901</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>16104</td>
<td>&nbsp;</td>
<tr>
<td>Electric pulse generator</td>
<td>Tool</td>
<td>Igea</td>
<td>&nbsp;</td>
<td>Cliniporator VITAE</td>
</tr>
<tr>
<td>Multiwell plate</td>
<td>Tool</td>
<td>TPP</td>
<td>92424</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91050</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91015</td>
<td>&nbsp;</td>
<tr>
<td>25 cm2 culture flask</td>
<td>Tool</td>
<td>TPP</td>
<td>90026</td>
<td>&nbsp;</td>
<tr>
<td>Electrodes</td>
<td>Tool</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
		</div>

cell electrofusion visualized with fluorescence microscopy

셀 electrofusion 인간의 치료를위한 하이브리드 전지를 준비 사용할 수있는 안전하고 비 바이러스성 및 비 - 화학 방법입니다. Electrofusion 가까운 접촉에있는 세포에 짧은 고전압 전기 펄스의 응용 프로그램을 포함합니다. 짧은 고전압 전기 펄스의 응용 프로그램 세포 플라즈마 점막의 불안정을 초래합니다. 불안 정한 점막은 다른 분자에 대한 더 투과하고 이웃 불안 정한 세포막과 융합하는 것도 쉽습니다. Electrofusion 따라서 매우 다른 세포가 아닌 구체적인 융합을 달성하기 위해 편리한 방법입니다<em&gt; 체외에서</em&gt;. 전기장에 의해 불안 정한 융합, 세포 세포막을 얻기 위해서는 그들의 지질 bilayers의 합병과 그 결과 그들의 세포질 수 있도록 가까운 접촉에 있어야합니다. 이 비디오에서는, 우리는 세포의 효율적인 electrofusion을 보여주<em&gt; 체외에서</em&gt; 수정된 준수 방법을 통해서 이루어집니다. 이 방법에서는, 세포가 잘의 표면에만 약간 첨부할 수 있으며, 매체는 교환 및 세포가 여전히 자신의 구형 형태를 유지 할 수 있도록. 퓨전 시각화 서로 다른 형광 셀 추적기 염료와 함께 세포의 세포질 사전 라벨 부착에 의해 평가되며 세포의 절반은 오렌지 CMRA 녹색 CMFDA과 나머지 절반으로 표시됩니다. 퓨전 수율은 두 곱한 모든 세포의 숫자로 나누어 dually 형광 세포의 숫자로 결정됩니다.

Watch this Scientific Journal Video about Cell Electrofusion Visualized with Fluorescence Microscopy at JoVE.com

Cell Electrofusion Visualized with Fluorescence Microscopy

이 비디오에서 우리는 세포의 효율적인 electrofusion을 보여주<em&gt; 체외에서</em&gt; electroporation과 형광 현미경과 세포 융합 시각화의 후속 감지를 사용하여 수정된 준수 방법을 통해서 이루어집니다.

세포는 형광 현미경 Electrofusion와 시각

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

cell-electrofusion-visualized-with-fluorescence-microscopy

Research

JoVE Journal

Biology

14.0K 조회수.  University of Ljubljana. 이 비디오에서 우리는 세포의 효율적인 electrofusion을 보여주 체외에서 electroporation과 형광 현미경과 세포 융합 시각화의 후속 감지를 사용하여 수정된 준수 방법을 통해서 이루어집니다.

비디오: Cell Electrofusion Visualized with Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

형광 현미경과 함께 머릿속으로 단일 분자의 DNA 복제

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

연구

생물학

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

개인 Presynaptic 터미널에서 도파민 릴리스 FFNs와 시각

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

분열 효모의 양적 라이브 세포 형광 - 현미경 분석

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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 ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

단일 비리온 원자력 현미경 검사및 초분해성 형광 이미징을 위한 샘플 준비

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

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 ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

라이브 세포 형광 현미경 검사 법 미생물 세포 성장 하는 동안 필수 과정을 관찰 하

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...