이 연구는 연방 교육 연구부 (FKZ 0312138A 및 FKZ 316159), 유럽 연합 구조 기금 (ESF / IVWM-B34-0030 / 10 및 ESF / IVBM-B35-0010 / 12), DFG (DA1296-1) 및 독일 심장 재단 (F / 01 / 12). 또한 RD는 Rostock University Medical Center (889001)의 FORUN 프로그램, DAMP Foundation 및 BMBF (VIP + 00240)의 지원을받습니다.

신생아 생쥐가 관련된이 프로토콜의 모든 단계는 Rostock University Medical Center의 동물 관리 지침에 따라 수행되었습니다. 1. 세포 배양 용 접시의 준비 및 심근 세포 배양 용 배지 <ol><li> PBS에서 0.1 % 젤라틴으로 세포 배양 플레이트를 코팅하고 37 ° C에서 4 시간 또는 4 ° C 밤새 인큐베이션하십시오. 젤라틴을 제거하고 멸균 층류 공기 흐름 하에서 건조 시키십시오. </li><li> 10 % 태아 소 혈청 (FBS)과 1 % 페니실린 / 스트렙토 마이신 (P / S)가 보충 된 DMEM 50 ML로 구성된 세포 배양 배지를 준비합니다. 37 ° C로 예열하십시오. </li></ol> 2. 신생아 심근 세포의 분리 <ol><li> 멸균 가위로 목을 베고 신생아 생쥐 (1 ~ 2 일 된)를 희생시키고 흉골을 따라 가슴을 엽니 다. 포어를 사용하여 가슴을 약간 제거하면서 가슴을 제거하고 심장을 얼음처럼 차가운 HBSS가 들어있는 24-well plate에 옮긴다.(Ca 2+ 및 Mg 2+ 없이). </li><li> 살균 포셉을 사용하여 비 심장 조직 및 큰 혈관을 제거합니다. 얼음처럼 차가운 HBSS가 들어있는 1.5 ML 튜브로 청소 마음을 전송하십시오. 효소 소화에는 튜브 당 최대 5 개의 하트를 사용하십시오. </li><li> 작은 가위로 가슴을 0.5-1mm³ 이하로 말하십시오. </li><li> 다량의 심장을 HBSS로 2 회 씻어 낸다. 1 mL microliter 피펫을 사용하여 HBSS를 흡입 / 첨가한다. 효소를 첨가하기 전에 HBSS를 완전히 제거하십시오. </li><li> 신생아 심장의 효소 소화를 위해서는 시중에서 구입할 수있는 키트를 사용하고 제조업체의 프로토콜 ( Table of Materials 참조)을 따르십시오. 35 분 동안 37 ° C에서 효소와 함께 잠복하면서 매 5 분마다 다진 하트가 담긴 튜브를 흔든다. </li><li> 비 - cardiomyocyte 분수의 준수를 허용 1.5-2 시간 세포 배양 배지를 포함하는 비 코팅 세포 배양 접시에 종자 세포를 중단. 높은 순도라면이 단계를 반복하십시오.의 심근 세포가 필요합니다. 원하는 경우, 비 - cardiomyocyte 분수를 추가로 배양하십시오. 참고 : 15 하트의 세포 현탁액의 경우, 75 cm² 세포 배양 플라스크에서 사전 도금 단계를 수행 할 수 있습니다. 일반적으로 한 신생아 심장에서 ~ 5 x 10 5 세포를 얻습니다. </li><li> 300 x g에서 10 분간 15 mL 원추형 튜브와 원심 분리기에서 뜨는 물을 수집합니다. 세포 배양 매체 1 ML에 세포를 Resuspend. </li><li> Neubauer 챔버 hemocytometer로 세포를 계산하거나, 동등한 방법을 사용하십시오. </li><li> 플레이트는 3 x 10 5 cells / cm²의 밀도를 지닌 6- 웰 플레이트상의 분리 된 심근 세포를 분리 하였다. 37 밤새 세포를 품어 ° C 5 % CO 2 . 참고 : 선택적으로이 시점에서 세포를 트랜 스펙 션 할 수도 있습니다. 그러나 일주일 동안 배양 한 후 일렉트로 포 레이션 (electroporation)을 실시 할 때 증가 된 생존력이 관찰되었다. </li></ol> 3. Fluorescently Labeled miRNA를 사용한 형질 감염 <ol><li> 사전 -따뜻한 세포 배양 배지 (단계 1.2 참조)를 수 욕조 또는 이와 동등한 장치에서 37 ° C로 가열합니다. </li><li> RNase가없는 멸균 수에서 형광성 miRNA의 20 μM 축적 용액을 준비하십시오. </li><li> 90 MM Na 2 HPO 4 , 90 MM NaH 2 PO 4 , 5 MM KCl, 10 MM MgCl 2 및 10 MM sodium succinate가 들어있는 일렉트로 포 레이션 버퍼를 준비하고 7.2로 pH를 조절하십시오. electroporation 버퍼는 -20 ° C에서 몇 개월 동안 저장할 수 있습니다. </li><li> 5 분 0.05 % 트립신을 사용하여 문화 요리에서 세포를 분리하십시오. 세포 배양 배지를 첨가하여 트립신을 비활성화시킨다. </li><li> Neubauer 챔버 hemocytometer로 세포를 계산하거나, 동등한 방법을 사용하십시오. 10 분 동안 300 XG에서 세포를 원심 분리기. </li><li> 100 μL 당 4 X 10 5 세포의 농도를 얻기 위해 일렉트로 포 레이션 버퍼에 세포를 Resuspend. </li><li> 튜브에 형광 miRNA (최종 농도 : 0.25 μm의)와 세포 현탁액의 100 μL를 혼합하고혼합물을 전기 천공 큐벳에 넣는다. <ol><li> 세포 유형에 따라 적절한 양의 miRNA를 경험적으로 결정하십시오. </li></ol></li><li> &quot;G - 009&quot;프로그램을 사용하여 일렉트로 포 레이션 장치 (물질 표 참조)를 사용하여 일렉트로 포 레이션을 수행하십시오. </li><li> 사전 예열 세포 배양 매체 500 μL를 추가하고 4 - 웰 유리 바닥 챔버 슬라이드의 우물에 전체 세포 현탁액 (4 X 10 5 세포)을 전송합니다. 5 % CO 2 분위기에서 37 ° C에서 하루 동안 세포를 배양하십시오. 참고 : FRAP 분석의 경우 ~ 80 %의 셀 밀도가 최적입니다. 분류 된 miRNA의 transfection은 electroporation을 사용하여 독점적으로 수행해야합니다. 분류 된 miRNA 분자의 균질 한 분포가 FRAP 측정에 유리하므로, 시약 기반 형질 감염은 권장하지 않습니다. </li></ol> 4. 3D-FRAP 현미경 검사 (3 일) 적용 <ol><li> 현미경 배양기를 37 °로 예열하십시오.C와 열 평형을 확립하고 표류의 가능성을 줄이기 위해 FRAP 측정 전에 적어도 2 시간 공 촛점 현미경 시스템을 전환합니다. 가능하면 5 % CO2 대기를 유지하십시오. </li><li> 스테이지 슬라이드 홀더에 챔버 슬라이드를 삽입합니다. </li><li> 1.4 NA 오일 대물 렌즈 (400 배율)와 낮은 레이저 출력에서 ​​561 nm의 레이저 여기 광을 사용하여 570-680 nm의 검출 범위로 트랜 스펙 션 된 심근 세포의 클러스터를 찾으십시오. 참고 : FRAP 설정, 이미지 획득 및 분석의 정의는 현미경 전용 소프트웨어를 사용하여 수행했습니다 ( 재료 표 참조). </li><li> &quot;Setup Manager&quot;에서 &quot;z-Stack&quot;, &quot;Time Series&quot;, &quot;Bleaching&quot;및 &quot;Regions&quot;버튼을 활성화하십시오. </li><li> FRAP 매개 변수를 정의하십시오. <ol><li> &quot;획득 모드&quot;메뉴에서 &quot;프레임 크기&quot;를 설정하여 이미지 획득 설정을 정의하십시오4; 512 x 512, &quot;라인 단계&quot;를 1로, &quot;스캔 시간&quot;을 1 초 미만으로 설정하십시오. </li><li> &quot;Channels&quot;메뉴에서 레이저 파워, 오프셋 및 게인 설정을 조정하여 최소 레이저 여기에서 최대 형광을 얻습니다 ( 예 : 레이저 출력 : 1-5 %). 강도 채도를 피하기 위해 조정하십시오. &quot;핀홀 크기&quot;를 2 μm로 설정하십시오. </li><li> 다음으로 &quot;Regions&quot;메뉴에서 &quot;ROI drawing tool&quot;을 선택하고 커서를 사용하여 대상 셀, 참조 셀 및 배경 영역을 표시합니다. 필요한 경우, 표백을 위해 여러 표적 세포를 선택하십시오. </li><li> &quot;표백&quot;메뉴 ( 예 : 반복 : 9-14, 광 표백을위한 레이저 출력 : 100 %, 이미지 획득 간격 : 60 초,주기 : 15)에서 표백 설정을 조정하십시오. 3 회 초기 스캔 후 표백 시작을 정의하십시오. </li><li> &quot;z-Stack&quot;메뉴에서 셀의 두께에 따라 Z- 스택 획득을위한 한계를 정의하십시오. &quot;숫자 o 조정f z-layers &quot;를 12 - 15로 설정합니다. </li><li> FRAP 실험을 시작하고 형광 복구를 기록합니다. 참고 : FRAP 실험의 설정은 세포 유형, 형광 염료 및 현미경 시스템에 따라 다르므로 파일럿 실험을 수행하여 FRAP에 대한 최적 매개 변수를 결정하는 것이 가장 좋습니다. 표백은 초기 형광 강도를 50 % 이상 줄이는 데 충분해야합니다. 일반적으로 형광 복구는 고원 단계에 도달 할 때까지 30-60 초마다 기록해야합니다. </li></ol></li></ol> 5. 데이터 분석 <ol><li> 획득 한 z- 스택의 최대 투사를 생성하고 표백 된 표적 세포, 참조 세포 및 배경의 형광 강도 값을 얻습니다. 현미경 전용 소프트웨어를 사용하여 &quot;Processing&quot;→ &quot;Maximum intensity projection&quot;→ 파일 선택 → &quot;Apply&quot;를 클릭하십시오. <ol><li> 또는, 동급 이미지 분석 도구 ( <e예를 들어, ImageJ). </li></ol></li><li> 대상 세포, 배경 및 참조 세포의 모든 시점에서 형광 강도 데이터를 스프레드 시트에 복사합니다. <ol><li> 획득 과정에서 발생한 photobleaching을 수정하기 위해 대상 세포의 강도 값에서 참조 셀의 배경 강도와 강도를 뺍니다. 각 시점에 대한 배경 및 기준 셀 보정을 수행하십시오. </li><li> 표백 전 초기 형광 강도로 각 값을 나눔으로써 보정 된 FRAP 데이터를 초기 형광 강도로 표준화하십시오. </li><li> FRAP 곡선을 얻으려면 각 시점의 강도 값에서 빼서 표백 직후의 형광 강도로 기준선을 설정하십시오. </li></ol></li></ol>

<ol>
	<li>Carthew, R. W., Sontheimer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+Mechanisms+of+miRNAs+and+siRNAs.">Origins and Mechanisms of miRNAs and siRNAs.</a> Cell. 136 (4), 642-655 (2009).</li><li>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391 (6669), 806-811 (1998).</li><li>Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+silencing+RNAs:+state-of-the-art.">Small silencing RNAs: state-of-the-art.</a> Adv Drug Deliv Rev. 61 (9), 672-703 (2009).</li><li>Raghunathan, S., Patel, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+implications+of+small+interfering+RNA+in+cardiovascular+diseases.">Therapeutic implications of small interfering RNA in cardiovascular diseases.</a> Fundam Clin Pharmacol. 27 (1), 1-20 (2013).</li><li>Sluijter, J. P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+Cardiovascular+Regenerative+Medicine:+Directing+Tissue+Repair+and+Cellular+Differentiation.">MicroRNAs in Cardiovascular Regenerative Medicine: Directing Tissue Repair and Cellular Differentiation.</a> ISRN Vasc Med. , 1-16 (2013).</li><li>Li, M., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+MicroRNAs:+Potential+and+Emerging+Biomarkers+for+Diagnosis+of+Cardiovascular+and+Cerebrovascular+Diseases.">Circulating MicroRNAs: Potential and Emerging Biomarkers for Diagnosis of Cardiovascular and Cerebrovascular Diseases.</a> Biomed Res Int. , 1-9 (2015).</li><li>Romaine, S. P. R., Tomaszewski, M., Condorelli, G., Samani, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+cardiovascular+disease:+an+introduction+for+clinicians.">MicroRNAs in cardiovascular disease: an introduction for clinicians.</a> Heart. 101 (12), 921-928 (2015).</li><li>Maes, M., Crespo Yanguas, ., Willebrords, S., Cogliati, J., Vinken, B., M, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+and+pannexin+signaling+in+gastrointestinal+and+liver+disease.">Connexin and pannexin signaling in gastrointestinal and liver disease.</a> Transl Res. 166 (4), 332-343 (2015).</li><li>Michela, P., Velia, V., Aldo, P., Ada, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+connexin+43+in+cardiovascular+diseases.">Role of connexin 43 in cardiovascular diseases.</a> Eur. J. Pharmacol. 768, 71-76 (2015).</li><li>Naus, C. C., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+and+challenges+of+connexin+connections+to+cancer.">Implications and challenges of connexin connections to cancer.</a> Nat Rev Cancer. 10 (6), 435-441 (2010).</li><li>Hong, X., Sin, W. C., Harris, A. L., Naus, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+modulate+glioma+invasion+by+direct+transfer+of+microRNA.">Gap junctions modulate glioma invasion by direct transfer of microRNA.</a> Oncotarget. 6 (17), 15566-15577 (2015).</li><li>Lee, H. K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+deliver+synthetic+microRNA+mimics+to+glioma+cells+and+glioma+stem+cells+and+inhibit+their+cell+migration+and+self-renewal.">Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal.</a> Oncotarget. 4 (2), 346-361 (2013).</li><li>Katakowski, M., Buller, B., Wang, X., Rogers, T., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+microRNA+is+transferred+between+glioma+cells.">Functional microRNA is transferred between glioma cells.</a> Cancer Res. 70 (21), 8259-8263 (2010).</li><li>Zong, L., Zhu, Y., Liang, R., Zhao, H. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction+mediated+miRNA+intercellular+transfer+and+gene+regulation:+A+novel+mechanism+for+intercellular+genetic+communication.">Gap junction mediated miRNA intercellular transfer and gene regulation: A novel mechanism for intercellular genetic communication.</a> Sci Rep. 6, 19884 (2016).</li><li>Yum, S. W., Zhang, J., Scherer, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+connexin26+mutants+associated+with+human+hearing+loss+have+trans-dominant+effects+on+connexin30.">Dominant connexin26 mutants associated with human hearing loss have trans-dominant effects on connexin30.</a> Neurobiol Dis. 38 (2), 226-236 (2010).</li><li>Kuzma-Kuzniarska, M., Yapp, C., Pearson-Jones, T. W., Jones, A. K., Hulley, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+gap+junctions+in+monolayer+and+three-dimensional+cultures+of+human+tendon+cells+using+fluorescence+recovery+after+photobleaching.">Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching.</a> J Biomed Opt. 19 (1), 15001 (2013).</li><li>Lemcke, H., Nittel, M. -. L., Weiss, D. G., Kuznetsov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+differentiation+requires+a+biphasic+modulation+of+gap+junctional+intercellular+communication+caused+by+dynamic+changes+of+connexin43+expression.">Neuronal differentiation requires a biphasic modulation of gap junctional intercellular communication caused by dynamic changes of connexin43 expression.</a> Eur J Neurosci. 38 (2), 2218-2228 (2013).</li><li>Lemcke, H., Steinhoff, G., David, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctional+shuttling+of+miRNA+-+A+novel+pathway+of+intercellular+gene+regulation+and+its+prospects+in+clinical+application.">Gap junctional shuttling of miRNA - A novel pathway of intercellular gene regulation and its prospects in clinical application.</a> Cell Signal. 27 (12), 2506-2514 (2015).</li><li>Lemcke, H., Kuznetsov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+connexin43+in+the+EGF/EGFR+signalling+during+self-renewal+and+differentiation+of+neural+progenitor+cells.">Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells.</a> Cell Signal. 25 (12), 2676-2684 (2013).</li><li>Lemcke, H., Peukert, J., Voronina, N., Skorska, A., Steinhoff, G., David, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+3D-FRAP+microscopy+to+analyse+gap+junction-dependent+shuttling+of+small+antisense+RNAs+between+cardiomyocytes.">Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.</a> J. Mol. Cell. Cardiol. 98, 117-127 (2016).</li><li>Laupheimer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Migration+of+Subpopulations+of+Bone+Marrow+Cells+along+an+SDF-1+&#945;+and+ATP+Gradient.">Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1 &#945; and ATP Gradient.</a> Bone Marrow Res. , 1-10 (2014).</li><li>Zhang, S., Wang, Q., Liu, L., Hong, X., Zhang, Y., Tao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+enhance+the+antiproliferative+effect+of+microRNA-124-3p+in+glioblastoma+cells.">Gap junctions enhance the antiproliferative effect of microRNA-124-3p in glioblastoma cells.</a> J Cell Physiol Physiol. 230 (10), 2476-2488 (2015).</li><li>Vasilescu, C., Tanase, M., Dragomir, M., Calin, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+mobility+to+crosstalk.+A+model+of+intracellular+miRNAs+motion+may+explain+the+RNAs+interaction+mechanism+on+the+basis+of+target+subcellular+localization.">From mobility to crosstalk. A model of intracellular miRNAs motion may explain the RNAs interaction mechanism on the basis of target subcellular localization.</a> Math Biosci. 280, 50-61 (2016).</li><li>Pitchiaya, S., Androsavich, J. R., Walter, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+single+molecule+microscopy+reveals+two+kinetically+distinct+pathways+for+microRNA+assembly.">Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly.</a> EMBO Rep. 13 (8), 709-715 (2012).</li><li>Heinze, K. G., Costantino, S., De Koninck, P., Wiseman, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+photobleaching,+laser+illumination+unbinds+fluorescent+proteins.">Beyond photobleaching, laser illumination unbinds fluorescent proteins.</a> J Phys Chem B. 113 (15), 5225-5233 (2009).</li><li>Jou, M. J., Jou, S. B., Guo, M. J., Wu, H. Y., Peng, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reactive+oxygen+species+generation+and+calcium+increase+induced+by+visible+light+in+astrocytes.">Mitochondrial reactive oxygen species generation and calcium increase induced by visible light in astrocytes.</a> Ann N Y Acad Sci. 1011, 45-56 (2004).</li><li>Dong, H., Lei, J., Ding, L., Wen, Y., Ju, H., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA:+Function,+detection,+and+bioanalysis.">MicroRNA: Function, detection, and bioanalysis.</a> Chem Rev. 113 (8), 6207-6233 (2013).</li><li>Liang, Y., Ridzon, D., Wong, L., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microRNA+expression+profiles+in+normal+human+tissues.">Characterization of microRNA expression profiles in normal human tissues.</a> BMC Genomics. 8 (1), 166 (2007).</li><li>Mittelbrunn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unidirectional+transfer+of+microRNA-loaded+exosomes+from+T+cells+to+antigen-presenting+cells.">Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.</a> Nat Commun. 2, 282 (2011).</li><li>Squadrito, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+RNAs+Modulate+MicroRNA+Sorting+to+Exosomes+and+Transfer+to+Acceptor+Cells.">Endogenous RNAs Modulate MicroRNA Sorting to Exosomes and Transfer to Acceptor Cells.</a> Cell Rep. 8, 1432-1446 (2014).</li><li>Zhang, B., Farwell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNAs:+a+new+emerging+class+of+players+for+disease+diagnostics+and+gene+therapy.">microRNAs: a new emerging class of players for disease diagnostics and gene therapy.</a> J Cell Mol Med. 12 (1), 3-21 (2008).</li><li>Chen, Y., Gao, D. -. Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery+of+miRNAs+for+cancer+therapy:+Challenges+and+strategies.">In vivo delivery of miRNAs for cancer therapy: Challenges and strategies.</a> Adv Drug Deliv Rev. 81C, 128-141 (2015).</li></ol>

저자는 아무런 이해 상충을 선언하지 않습니다.

<html> miRNA는 세포 생리학에서 중요한 역할을 담당하며, 다른 것들 중에서도 GJ를 세포 간 교환을위한 경로 11 , 12 , 22 로 사용함으로써 신호 분자로 작용하는 것으로 나타났습니다. 현재 프로토콜 세포 클러스터 내에서 형광 miRNAs를 사용 하여이 GJ 의존 shuttling을 특성화 하는 체외 라이브 셀 이미징 기술을 제공합니다. <...

작은 noncoding RNA는 세포 유전자 조절에서 중요한 역할을합니다. 이 분자는 특정 표적 mRNA에 결합하는 20-25 개의 뉴클레오타이드로 이루어져 번역의 막힘 또는 mRNA 분해 1 또는 2를 유도합니다. miRNA와 siRNA와 같은 작은 RNA에 의해 수행되는 유전자 조절 과정은 많은 다른 종에서 발견 된 매우 보전 된 메커니즘입니다 3 . 특히, miRNA 분자는 증식, 분화 및 재생을 포함한 다양한 생리적 과정에 매우 중요합니다. 또한, miRNA 발현의 조절 장애는 많은 병리학 적 장애로 인한 것입니다. 그에 상응하여, miRNA는 진단을위한 바이오 마커 및 유전자 요법을위한 치료제로서 적합하다는 것이 입증되었습니다 6 , 7 </sup&gt;. GJ는 분자량이 1kD 인 분자를 확산 교환 할 수있는 인접한 두 세포의 원형질막에 특화된 단백질 구조입니다. 그들은 조직 발달, 분화, 세포 사멸, 암이나 심혈관 질환과 같은 병리학 적 장애에 중요한 것으로 밝혀졌습니다 8 , 9 , 10 . 여러 분자가 이온, 대사 산물 및 뉴클레오타이드를 포함하여 GJ 채널을 통과 할 수 있다고 기술되어 왔습니다. 흥미롭게도 GJ는 작은 RNA 11,12 의 세포 간 이동을위한 경로를 제공하는 것으로 밝혀졌습니다. 따라서, miRNAs는 생산 된 세포 내에서뿐만 아니라 수용 세포 내에서 작용할 수 있습니다. 이것은 세포 간 신호 전달 시스템에서 miRNA의 역할을 강조합니다. 동시에, 데이터는그 gap junctional intercellular communication은 miRNA 기능과 밀접하게 연관되어있다. 조직 항상성, 병리학 및 진단에 대한 miRNA 및 GJ의 중요한 영향으로 인해 GJ의 기능과 miRNA의 관련 세포 간 역학에 대한 포괄적 인 이해는 miRNA 기반 질병의 메커니즘을 명확히하고 새로운 전략을 개발하는 데 도움이 될 것입니다. miRNA 치료법. gap junctional coupling의 정도에 따라 세포 간 miRNA 분자의 이동은 매우 신속한 과정이 될 수 있습니다. 따라서, 이들 조절 신호 분자의 빠른 세포 간 이동의 시각화 및 정량화를 가능하게하는 방법론이 요구된다. 일반적으로 작은 RNA 11 , 12 , 13 , 14 의 왕복을 입증하기 위해 유동 세포 계측법과 분자 생물학적 기술이 적용되었습니다. 그러나 FRAP 마이크로와는 달리이러한 접근법은 GJ를 통한 miRNA 교환을 분석 할 때 높은 시간 해상도가 필요하지 않습니다. 더욱이 FRAP 현미경 검사법은 덜 침습적이므로 여러 세포 유형에서 GJ에 의존하는 분자의 교환을 평가하는 강력하고 새로운 생체 ​​영상 기술입니다 15 , 16 , 17 . 여기, 우리는 cardiomyocytes 사이에 miRNA 셔틀을 평가하는 3D - FRAP의 응용 프로그램을 설명하는 자세한 프로토콜을 제시한다. 이 목적을 위해, 형광성으로 표지 된 miRNA로 심근 세포를 형질 감염시켰다. 이 miRNA로 표시된 세포는 광 표백되었고, 인접한 세포로부터 다시 유입되는 gap junctional miRNA는 시간 의존적으로 기록되었다. FRAP 실험의 높은 시간 해상도는 살아있는 세포 사이의 miRNA 및 siRNA의 세포 간 전달을 정확하게 평가하기위한 운동 연구를 수행 할 수있는 가능성을 제공합니다. 모레 오ver., 작은 RNA는 매우 다른 동력학을 가진 다른 메커니즘을 통해 교환 될 수 있기 때문에, FRAP 현미경 검사는 GJ가 각각의 왕복 운동 과정에 얼마나 관여 하는지를 명확히하는 데 도움이 될 수 있습니다. 또한 3D-FRAP은 GJ 투과성의 생리 학적 및 병리학 적 변화와 작은 RNA 전이에 미치는 영향을 조사하는 데 사용할 수 있습니다 15 , 19 .

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">neonatal NMRI mice</td>
			<td style="width:159px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Gelatin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">G7041</td>
			<td style="width:179px;">0.1% solution in PBS, sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PBS</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-53500</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Dulbecco&#180;s modified medium</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-03550</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin/Streptomycin</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140122</td>
			<td style="width:179px;">100 U/ml, 100&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal bovine serum</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P30-3306</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Cell culture plastic</td>
			<td style="width:159px;">TPP</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Primary Cardiomyocyte Isolation Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">HBSS</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;">included in primary cardiomyocyte isolation kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypan blue</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">miRIDIAN Dy547 labeled microRNA Mimic&#160;</td>
			<td style="width:159px;">Dharmacon</td>
			<td style="width:119px;">CP-004500-01-05</td>
			<td style="width:179px;">dissolve in RNAse free water, stock solution 20&#181;M</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:243px;">Sodium phosphate monobasic</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">S3139</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium phosphate dibasic</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">T877.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium chloride</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Magnesium chloride</td>
			<td style="width:159px;">Serva</td>
			<td style="width:119px;">28305.01</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium succinate</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">3195.1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin/EDTA solution</td>
			<td style="width:159px;">Merck</td>
			<td style="width:119px;">L2153</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">4-well- Glass bottom chamber slides</td>
			<td style="width:159px;">IBIDI</td>
			<td style="width:119px;">80827</td>
			<td style="width:179px;">coat with 0.1% gelatin solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Amaxa Nucleofector II</td>
			<td style="width:159px;">Lonza</td>
			<td style="width:119px;"></td>
			<td style="width:179px;">program G-009 was used for Electroporation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LSM 780 ELYRA PS.1 system</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Excel software</td>
			<td style="width:159px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#945;-actinin antibody</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:119px;">ab9465</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Connexin43 antibody</td>
			<td style="width:159px;">Santa Cruz</td>
			<td style="width:119px;">sc-9059</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-mouse Alexa 594 antibody</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11005</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-rabbit Alexa 488 antibody&#160;</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11034</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Connexin43 siRNA</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM16708 ID158724</td>
			<td>&#160;final concentration 250nM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Near-IR Live/Dead Cell Stain Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CellTrace Calcein Red-Orange</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DAPI nuclear stain</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy

miRNA 및 siRNA와 같은 작은 안티센스 RNA는 세포 생리학 및 병리학에서 중요한 역할을하며, 또한 여러 질병의 치료에서 치료제로 사용될 수 있습니다. miRNA / siRNA 치료를위한 새롭고 혁신적인 전략의 개발은 근본적인 메커니즘에 대한 광범위한 지식을 기반으로합니다. 최근의 데이터는 작은 RNA가 갭 접합부 (gap junction-dependent) 방식으로 세포들 사이에서 교환 됨으로써 수용 세포에서 유전자 조절 효과를 유도 함을 시사한다. 분자 생물학적 기법과 유동 세포 계측 분석은 일반적으로 miRNA의 세포 간 교환을 연구하는 데 사용됩니다. 그러나, 이러한 방법은 높은 시간 해상도를 제공하지 못하며, 이것은 분자의 갭 접합 플럭스를 연구 할 때 필요하다. 따라서 miRNA / siRNA가 세포 간 신호 전달 분자로서의 영향을 조사하기 위해서는 이러한 작은 RNA를 세포 수준에서 분석 할 수있는 새로운 도구가 필요합니다. 현재의 프로토콜은 ap(3 차원 - FRAP) 현미경 검사 후 3 차원 형광 복구의 plication은 심장 세포 사이의 갭 접합 - 의존적 인 miRNA 분자의 교환을 해명합니다. 중요한 것은,이 간단하고 비 침습적 인 라이브 셀 이미징 접근 방식은 높은 시공간 해상도로 실시간으로 형광 표지 된 작은 RNA의 갭 접합 셔틀 링의 시각화 및 정량화를 허용합니다. 3D-FRAP에서 얻은 데이터는 작은 RNA가 세포 간 네트워크 내에서 신호 분자로 작용하는 세포 간 유전자 조절의 새로운 경로를 확인합니다.

여기, 신생아 cardiomyocytes 이내 형광 miRNA의 간격 junctional 셔틀을 연구 비 침습적 인 기술로 3D - FRAP 현미경을 제시합니다. 분리 된 심근 세포는 전형적으로 흘린 α- 액티 닌 패턴을 나타내었고, 세포 간 경계 ( 그림 1A , 흰 화살촉)를 따라 Cx43의 큰 플라크를 포함하여 분자간의 높은 세포 간 플럭스를 허용했다. 단리 된 심근 세포의 순도는 α-actinin 양성 ?...

Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

여기, 우리는 miRNA의 갭 접합 종속 셔틀 (gap junction-dependent shuttling) 분석을위한 광 표백 (photobleaching) 후 3 차원 형광 복구 (3D-FRAP)의 응용을 기술한다. 일반적으로 적용되는 방법과는 달리, 3D-FRAP은 높은 시공간 해상도로 실시간으로 작은 RNA의 세포 간 전달을 정량화합니다.

3D-FRAP 현미경을 이용한 miRNA의 gap junction 의존적 전달 분석

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

analysis-gap-junction-dependent-transfer-mirna-with-3d-frap

Research

JoVE Journal

Biology

8.8K 조회수.  Reference and Translation Center for Cardiac Stem Cell Therapy (RTC).  여기, 우리는 miRNA의 갭 접합 종속 셔틀 (gap junction-dependent shuttling) 분석을위한 광 표백 (photobleaching) 후 3 차원 형광 복구 (3D-FRAP)의 응용을 기술한다. 일반적으로 적용되는 방법과는 달리, 3D-FRAP은 높은 시공간 해상도로 실시간으로 작은 RNA의 세포 간 전달을 정량화합니다. 

비디오: Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

주변 줄기 세포 이주의 분석을위한 마우스 Cremaster 근육에있는 미세 혈관의의 intravital 현미경

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

연구

생물학

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

<em&gt; Xenopus의</em형광 측정으로&gt; 난자 절개 바셀린 갭 전압 클램프 기술

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

신속한 분석 및 형광 현미경 이미지의 탐사

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

자식 작용의 분석에<em&gt; 페니 chrysogenum</em&gt; 형광 현미경과 조합 기아 패드를 사용하여

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFE의 분석을 사용하여 높은 처리량 miRNA의 표적의 탐지

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

가변 각도 표면 형광 현미경으로 식물 세포 표면 역학의 실시간 영상

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapper 슈퍼 해상도 현미경을위한 오픈 소스 단일 입자 분석

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

고압 냉동, 전자 레인지를 이용한 콘트라스트 향상, 그리고 볼륨 이미지에 대 한 포함 하는 최소한의 수 지에 의해 생물학 견본 준비

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

총 내부 반사 형광 현미경 검사법에 의해 살아있는 세포에 있는 세포 표면 수용체의 올리고머화 역학 은 수 및 밝기 분석과 결합됩니다

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

금 나노 입자와 지질 막 사이의 열 전달을 모니터링하는 테더링 된 바이 레이어 지질 멤브레인

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

3D-FRAP 현미경을 이용한 miRNA의 gap junction 의존적 전달 분석

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