We thank Stephan Uphoff for discussions. 
R.C. was supported by Linacre College, Oxford University. A.P. was supported by the German Academic Exchange Service (DAAD), the German National Academic Foundation and EPSRC. M.S. was supported by the Wellcome Trust. A.N.K. was supported by a UK BBSRC grant (BB/H01795X/1), and a European Research Council Starter grant (261227).

<ol>
	<li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu Rev Biochem. 67, 509-544 (1998).</li><li>Leake, M. C., 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=Stoichiometry+and+turnover+in+single,+functioning+membrane+protein+complexes.">Stoichiometry and turnover in single, functioning membrane protein complexes.</a> Nature. 443, 355-358 (2006).</li><li>Taniguchi, Y., Kawakami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+HaloTag+protein+to+covalent+immobilization+of+recombinant+proteins+for+single+molecule+force+spectroscopy.">Application of HaloTag protein to covalent immobilization of recombinant proteins for single molecule force spectroscopy.</a> Langmuir. 26, 10433-10436 (2010).</li><li>Xie, X. S., Choi, P. J., Li, G. W., Lee, N. K., Lia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+approach+to+molecular+biology+in+living+bacterial+cells.">Single-molecule approach to molecular biology in living bacterial cells.</a> Annual review of biophysics. 37, 417-444 (2008).</li><li>Lee, J. 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=Highly+multiplexed+subcellular+RNA+sequencing+in+situ.">Highly multiplexed subcellular RNA sequencing in situ.</a> Science. 343, 1360-1363 (2014).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Sauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+microscopy+coming+of+age:+from+concepts+to+biological+impact.">Localization microscopy coming of age: from concepts to biological impact.</a> J Cell Sci. 126, 3505-3513 (2013).</li><li>Dempsey, G. T., Vaughan, J. C., Hao Chen, K., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorophores+for+optimal+performance+in+localizationbased+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localizationbased super-resolution imaging.</a> Nat Meth. 8, 1027-1041 (2011).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Meth. 2, 905-909 (2005).</li><li>Landgraf, D., Okumus, B., Chien, P., Baker, T. A., Paulsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+molecules+at+cell+division+reveals+native+protein+localization.">Segregation of molecules at cell division reveals native protein localization.</a> Nat. Methods. 9, 480-482 (2012).</li><li>Jaitin, D. A., 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=Massively+Parallel+Single-Cell+RNA-Seq+for+Marker-Free+Decomposition+of+Tissues+into+Cell+Types.">Massively Parallel Single-Cell RNA-Seq for Marker-Free Decomposition of Tissues into Cell Types.</a> Science. 343, 776-779 (2014).</li><li>Aldridge, S., 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=AHT-ChIP-seq:+a+completely+automated+robotic+protocol+for+high-throughput+chromatin+immunoprecipitation.">AHT-ChIP-seq: a completely automated robotic protocol for high-throughput chromatin immunoprecipitation.</a> Genome Biol. 14, R124 (2013).</li><li>Keppler, A., 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=A+general+method+for+the+covalent+labeling+of+fusion+proteins+with+small+molecules+in+vivo.">A general method for the covalent labeling of fusion proteins with small molecules in vivo.</a> Nat Biotechnol. 21, 86-89 (2003).</li><li>Wombacher, R., 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=Live-cell+super-resolution+imaging+with+trimethoprim+conjugates.">Live-cell super-resolution imaging with trimethoprim conjugates.</a> Nat. Methods. 7, 717-719 (2010).</li><li>Zhang, Z., 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=A+new+strategy+for+the+site-specific+modification+of+proteins+in+vivo.">A new strategy for the site-specific modification of proteins in vivo.</a> Biochemistry. 42, 6735-6746 (2003).</li><li>McNeil, P. L., Murphy, R. F., Lanni, F., Taylor, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+incorporating+macromolecules+into+adherent+cells.">A method for incorporating macromolecules into adherent cells.</a> J Cell Biol. 98, 1556-1564 (1984).</li><li>Clarke, M. S., McNeil, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syringe+loading+introduces+macromolecules+into+living+mammalian+cell+cytosol.">Syringe loading introduces macromolecules into living mammalian cell cytosol.</a> J Cell Sci. 102, 533-541 (1992).</li><li>Sakon, J. J., Weninger, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+the+conformation+of+individual+proteins+in+live+cells.">Detecting the conformation of individual proteins in live cells.</a> Nat. Methods. 7, 203-205 (2010).</li><li>Taylor, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromagnetic+syringe.">Electromagnetic syringe.</a> IEEE Trans. Biomed. Eng. 25, 303-304 (1978).</li><li>Dower, W. J., Miller, J. F., Ragsdale, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+E.+coli+by+high+voltage+electroporation.">High efficiency transformation of E. coli by high voltage electroporation.</a> Nucleic Acids Res. 16, 6127-6145 (1988).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-845 (1982).</li><li>Sustarsic, 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=Optimized+delivery+of+fluorescently+labeled+proteins+in+live+bacteria+using+electroporation.">Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation.</a> Histochem Cell Biol. , (2014).</li><li>Tokunaga, M., Imamoto, N., Sakata-Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+inclined+thin+illumination+enables+clear+single-molecule+imaging+in+cells.">Highly inclined thin illumination enables clear single-molecule imaging in cells.</a> Nat. Methods. 5, 159-161 (2008).</li><li>Sinha, A., 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=A+cascade+of+DNA-binding+proteins+for+sexual+commitment+and+development+in+Plasmodium.">A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.</a> Nature. 000, 1-5 (2014).</li><li>English, B. P., 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=Single-molecule+investigations+of+the+stringent+response+machinery+in+living+bacterial+cells.">Single-molecule investigations of the stringent response machinery in living bacterial cells.</a> Proc Natl Acad Sci U S A. 108, E365-E373 (2011).</li><li>Crawford, R., 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=Long-lived+intracellular+single-molecule+fluorescence+using+electroporated+molecules.">Long-lived intracellular single-molecule fluorescence using electroporated molecules.</a> Biophys J. 105, 2439-2450 (2013).</li><li>Uphoff, S., Reyes-Lamothe, R., Garza de Leon, F., Sherratt, D. J., Kapanidis, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+DNA+repair+in+live+bacteria.">Single-molecule DNA repair in live bacteria.</a> Proc Natl Acad Sci U S A. 110, 8063-8068 (2013).</li><li>Uphoff, S., Sherratt, D. J., Kapanidis, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Protein-DNA+Interactions+in+Live+Bacterial+Cells+Using+Photoactivated+Single-molecule+Tracking.">Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking.</a> J Vis Exp. , (2014).</li><li>Hohlbein, J., Gryte, K., Heilemann, M., Kapanidis, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfing+on+a+new+wave+of+single-molecule+fluorescence+methods.">Surfing on a new wave of single-molecule fluorescence methods.</a> Phys Biol. 7, 031001 (2010).</li><li>Xie, X. S., Yu, J., Yang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspective+-+Living+cells+as+test+tubes.">Perspective - Living cells as test tubes.</a> Science. 312, 228-230 (2006).</li><li>Santoso, Y., 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=Conformational+transitions+in+DNA+polymerase+I+revealed+by+single-molecule+FRET.">Conformational transitions in DNA polymerase I revealed by single-molecule FRET.</a> Proc Natl Acad Sci U S A. 107, 715-720 (2010).</li><li>Hohlbein, J., 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=Conformational+landscapes+of+DNA+polymerase+I+and+mutator+derivatives+establish+fidelity+checkpoints+for+nucleotide+insertion.">Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion.</a> Nature communications. 4, 2131 (2013).</li></ol>

The authors have nothing to disclose.

De nombreux paramètres peuvent être modifiés pendant l&#39;électroporation des cellules et d&#39;acquisition de données selon le système biologique d&#39;intérêt et la nature précise de l&#39;expérience (au niveau de la cellule ou molécule unique analyse). Par exemple, lorsque l&#39;ADN par électroporation dans des bactéries, 0,25 à 5 pmoles de fragments d&#39;ADN double brin marqués conduit à une faible efficacité d&#39;internalisation, ce qui permet la détection directe seule molécule (par exemple., Sans avoir besoin de photoblanchiment à l&#39;avance). Au-dessus de 5 pmol ADN double brin, les cellules ont tendance à être lourdement chargé, un régime mieux adapté pour l&#39;analyse unicellulaire. Tous les ADN marqués devraient également être préalablement purifié sur gel pour éliminer toute trace de colorant libre (fluorophore ne ayant pas réagi) de la solution ADN stock. En outre, les problèmes potentiels avec la dégradation de l&#39;ADN, en particulier pour des expériences smFRET, peuvent être réglés par des ADN avec des acides nucléiques non naturels, ou des motifs qui protègent exonucléase accessible terminaisons tels que des boucles en épingle à cheveux. Un autre adjustable paramètre dans électroporation est l&#39;intensité du champ appliqué pendant l&#39;électroporation. Faible intensité de champ (~ 1 kV / cm) conduira à une faible efficacité de chargement approprié pour les études à molécule unique. Intensités de champ plus élevée (jusqu&#39;à 1,8 kV / cm) augmenteront l&#39;efficacité de chargement; Cependant, il existe une corrélation inverse entre l&#39;intensité du champ et la viabilité des cellules après l&#39;électroporation (voir figure 4). Pour référence, une intensité de champ normal utilisé pour les bactéries et la levure est une électroporation à environ 1,5 kV / cm. La constante de temps représentant la durée de cette décomposition, est un paramètre pratique à suivre, car les gouttes de constante de temps dès que tout phénomène d&#39;arc se produit dans la cuvette. D&#39;après les réglages normaux, la constante de temps doit être supérieure à 4 ms; des valeurs inférieures conduiront à une faible efficacité de chargement ou de cellules endommagées, même non-chargées. La plupart des électroporateurs offrent d&#39;autres degrés de liberté (comme &quot;troncature d&#39;impulsion» ou «forme des impulsions&quot;) qui peut être modifiée pour accorder à la foisle chargement et la viabilité cellulaire. Nous avons appliqué cette méthode à la fois les bactéries et les levures, cependant procédures similaires devraient également permettre l&#39;internalisation des biomolécules marquées dans les cellules de mammifère en utilisant les paramètres de électroporateur appropriées depuis leur membrane est en fait moins complexe (unique bicouche lipidique) et depuis l&#39;électroporation a déjà été utilisé avec de telles cellules 21. Lorsque l&#39;internalisation des protéines marquées, tout colorant libre doit être retiré de la solution de protéine marquée des stocks électroporation préalable. Molécules de colorant libres, en raison de leur petite taille, peuvent être internalisés préférentiellement au cours des protéines d&#39;intérêt, et sont difficiles à distinguer lors de l&#39;analyse des données (malgré leur diffusion devrait plus rapide). A titre indicatif, pour un échantillon biologique de la protéine marquée pour être adapté à l&#39;électroporation, la quantité de colorant restant libre doit être inférieure à 2% (balayage à fluorescence détectée à l&#39;aide d&#39;une SDS-PAGE) 22. Ce procédé est particulièrement importante,que certaines molécules pourraient tenir à des membranes externes des bactéries ou des levures par électroporation. À cet égard, l&#39;échantillon de contrôle négatif doit afficher intensité de fluorescence par cellule nettement plus faible que les cellules électroporées, idéalement aussi bas que le niveau de cellules vides (cellules qui ne ont pas été incubées avec des biomolécules marquées par fluorescence, ni électroporation, figure 2) autofluorescence. Comme avec l&#39;ADNdb, l&#39;efficacité d&#39;internalisation des protéines marquées est liée à la quantité de biomolécules ajouté aux cellules avant l&#39;électroporation. Cependant, d&#39;autres paramètres, comme la taille et la charge, jouent un rôle dans l&#39;internalisation. Les petites protéines présentent des rendements élevés d&#39;internalisation, tandis que les protéines plus grandes (jusqu&#39;à 98 kDa) peut être internalisé avec succès, mais avec une efficacité plus faible (figure 5) 26. Le point isoélectrique de la protéine, les interactions potentielles avec la membrane cellulaire et d&#39;autres paramètres physico-chimiques aussiinfluence chargement cellulaire pendant électroporation. En conséquence, les utilisateurs ont besoin pour optimiser les expériences de leur propre système, sachant qu&#39;une forte concentration initiale de protéine marquée (&gt; 50 M) donnera la meilleure chance pour le chargement réussi. Électroporation propose également un nouvel outil de perturber et d&#39;analyser la fonction cellulaire en introduisant des protéines et d&#39;autres biomolécules dans les cellules (soit marqué ou non marqué). Les expériences de la T7 ARN polymerase (Figure 5C) présentent une telle exemples d&#39;une expérience dans laquelle on peut introduire une biomolécule qui peut modifier l&#39;expression de gènes in vivo en utilisant l&#39;électroporation. Lorsque la réalisation d&#39;expériences de fluorescence seule molécule, illumination TIR est généralement privilégiée par rapport à d&#39;autres modes d&#39;éclairage car il offre le meilleur rapport signal-sur-bruit en excitant seulement fluorophores dans une section mince dessus de la surface de lamelle (~ 100 nm). Cependant imagerie de diffusion à l&#39;intérieur des biomolécules marquées micro-organismes vivants peuvent reexiger un éclairage plus profond (jusqu&#39;à 0,8 um pour E. coli). Deeper illumination est réalisée en mode Hilo, tout en préservant un rapport signal-bruit. D&#39;autre part, l&#39;imagerie à grand champ est particulièrement important pour l&#39;analyse de photoblanchiment étapes, où l&#39;utilisateur est l&#39;estimation du nombre de molécules internalisées par photoblanchiment toute une cellule chargée avec une puissance laser élevée et en divisant l&#39;intensité initiale de la fluorescence de la cellule par l&#39;intensité unitaire produite par une seule molécule (l&#39;étape de photoblanchiment unique, figure 3). Imagerie Widefield est également nécessaire pour le suivi de la molécule à long terme afin de localiser les molécules diffusantes d&#39;intérêt même si leurs trajectoires couvrent le volume de cellule entière. Dans ce protocole, nous présentons comment électroporation, une technique standard pour les biologistes et de biochimistes pour délivrer des acides nucléiques dans les cellules, constitue une méthode simple pour délivrer biomolécules fluorescentes dans divers types de cellules. Thest nouvelle technique à haut débit offre un outil unique d&#39;observer des molécules marquées dans leur environnement natif. En d&#39;addition biomolécules marquées avec des fluorophores couvrant une large gamme de longueurs d&#39;onde, l&#39;électroporation peut délivrer des molécules modifiées par de nombreux groupes chimiques, tels que des nucleotides et des acides aminés non naturels, des chélateurs de métaux, des agents de réticulation, et les groupes de mise en cage. Si le système biologique d&#39;intérêt ne est pas indispensable au développement de la cellule, le gène codant pour la protéine cible peut également être supprimée (ou knocked-down), se assurer que les protéines observées après internalisation représentent tous (ou presque) de la piscine de protéine intracellulaire . En substance, l&#39;électroporation peut &quot;greffe&quot; la flexibilité de bioconjugués in vitro dans des cellules vivantes et donc bénéficier des efforts de la biologie synthétique, la biologie des systèmes, et dans la détection de molécule unique vivo.

La plupart des études de fluorescence à l&#39;intérieur des cellules vivantes dépendent de fusions de protéines avec des protéines fluorescentes (MF), comme une GFP. Ces études permettent marqueurs fluorescents du nombre de copies, motif de diffusion ou la localisation des protéines impliquées dans des processus tels que l&#39;expression du gène ou du transport membranaire 2-7. MF offrent la spécificité d&#39;étiquetage haute, la mise en œuvre facile, et sont disponibles dans un grand inventaire des variantes avec divers photophysique et les propriétés chimiques 1. Cependant, fluorophores organiques restent le premier choix pour des expériences in vitro en raison de leur plus grande photostabilité (jusqu&#39;à 100 fois plus stable que MF) 8,9, de petite taille (jusqu&#39;à 100 fois plus petit volume que MF) et la facilité d&#39;étiquetage intramoléculaire (principalement par l&#39;utilisation de résidus de cysteine). Tous ces facteurs sont particulièrement importants pour la fluorescence seule molécule et FRET 10 études. Plusieurs méthodes d&#39;internalisation Combincompris les avantages de l&#39;étiquetage biologique et dans la détection in vivo ont été introduites au cours de la dernière décennie; Cependant, ces méthodes soit emploient relativement importantes polypeptides balises (tags par exemple, TMP, HALO, ou 20 kDa SNAP) 11-14, nécessitent l&#39;utilisation d&#39;acides aminés non naturels 15, ou sont limités à grandes cellules eucaryotes, seule membrane (par exemple. , gratter chargement, seringue chargement, la micro-injection) 16-19. Ce protocole décrit un roman, simple et à haut débit méthode d&#39;internalisation que les couples les avantages de fluorophores organiques avec l&#39;observation in vivo. Pour développer cette technique, nous avons adapté le procédé d&#39;électroporation couramment utilisé pour transformer des cellules avec l&#39;ADN plasmidique 20,21 afin de charger les micro-organismes tels que E. coli ou S. cerevisiae avec des biomolécules organiquement étiquetés. Le protocole se compose de quatre étapes simples: l&#39;incubation des cellules avec des biomolécules marquées,l&#39;électroporation, la récupération des cellules, et le lavage des cellules pour éliminer les biomolécules non-internalisé. Ici, nous présentons ce protocole d&#39;électroporation, ainsi que les procédés d&#39;imagerie cellulaire et d&#39;analyse de données pour étudier la fluorescence à base de cellules et une seule molécule et FRET signaux. L&#39;électroporation de décharge repose sur un champ électrique à haute tension à travers une faible ionique suspension de cellules de force pour former des pores de la membrane à travers laquelle transitoire biomolécules peuvent pénétrer dans les cellules (figure 1) 20,21. Tout comme avec la transformation de bactéries ou de la levure avec de l&#39;ADN plasmidique, les cellules doivent être préparés avant l&#39;électroporation pour assurer leur electrocompetency. Cette procédure, composé de plusieurs étapes de lavage avec de l&#39;eau, augmente la perméabilité de la membrane et réduit la force ionique de la solution de cellules pour éviter un arc électrique dans la cuvette d&#39;électroporation. Dans ce protocole, les cellules peuvent être préparés comme décrit ci-dessous (voir le protocole: 1.1) ou acheté d&#39;un fournisseur commercials. <img alt="Figure 1" src="/files/ftp_upload/52208/52208fig1.jpg" /> Figure 1: Représentation schématique du protocole d&#39;internalisation De gauche à droite:. Ajouter quelques microlitres de biomolécules marquées à l&#39;aliquote de cellules électrocompétentes (fragments d&#39;ADN doublement marquée et les bactéries dans cet exemple); incuber 1 à 10 minutes sur de la glace et à transférer une cuvette d&#39;électroporation de pré-refroidi; électroporer puis ajouter 0,5 à 1 ml de milieu riche pour les cellules immédiatement après; incuber à 37 ° C (ou la température requise par l&#39;organisme, par exemple, 29 ° C pour les levures) pour laisser les cellules se rétablir; effectuer des 5 étapes de lavage pour enlever l&#39;excédent non-internalisées molécules marquées; remettre en suspension le culot final dans 100-200 pi de tampon PBS et pipette 10 ul sur un tampon d&#39;agarose; couvrir le pad avec une lamelle nettoyé et image sur un microscope à fluorescence (en mode grand champ ou en mode HILO). <p class = &quot;jove_content&quot;&gt; cellules électrocompétentes sont incubées avec les biomolécules marqués juste avant électroporation, qui peuvent être effectuées à l&#39;aide électroporateurs standards trouvés dans la plupart des laboratoires de biochimie. Immédiatement après l&#39;électroporation, les cellules sont incubées dans un milieu riche qui permet leur récupération avant de les laver (Figure 1). L&#39;excès de biomolécules marquées non-internalisées est d&#39;abord éliminé par lavage dans un tampon contenant une concentration relativement élevée de sel et un peu de détergent (Voir PROTOCOLE: 3,3). La présence de sel perturbe interactions électrostatiques non spécifiques formés par des biomolécules marquées non internalisés qui autrement peuvent coller sur la membrane externe. De même, la présence de détergent dans le tampon de lavage perturbe interactions hydrophobes non spécifiques. Alors que l&#39;ADN internalisation est simple (Figure 2), des précautions doivent être prises lors de l&#39;internalisation des protéines marquées à l&#39;aide de l&#39;électroporation. Tout d&#39;abord, le stock échantillon de protéines organiquement marqué pourrait encore contenir un petit pourcentage de colorant libre. Molécules de colorant libres sont beaucoup plus petites que les protéines et pourraient donc être internalisés préférentiellement. Pour se assurer que la grande majorité des molécules fluorescentes internalisés observées correspondent à la protéine d&#39;intérêt, l&#39;échantillon de protéine initiale doit contenir moins de 2% de colorant ~ libre (Figure 5) 22. L&#39;excès de protéines marquées non-internalisées peut également tenir à la membrane cellulaire externe après l&#39;électroporation; ce phénomène est spécifique à la protéine et doit être vérifié pour chaque nouvelle protéine. Nous vous proposons plusieurs options qui permettent l&#39;élimination des protéines non-intériorisée de l&#39;échantillon de cellules chargé (Voir PROTOCOLE: 3.3.3). Enfin, les cellules sont remises en suspension dans un petit volume de tampon phosphate et un tampon pipette sur agarose, ce qui permet leur image sur un microscope à fluorescence. Immobilisation sur des patins agarose est un simple et moyen efficace de l&#39;imagerie des cellules sur une lamelle sans nuire à leur intégrité. Le tampon doit contenir un milieu de culture à faible fluorescence. Imagerie cellulaire peut être effectuée soit en champ large, totale fluorescence à réflexion interne (FRBR) ou en utilisant HILO (fortement inclinées et laminé feuille optique) microscopie. Dans la configuration HILO, le faisceau laser pénètre plus profondément dans l&#39;échantillon que dans TIRF, mais ne se allume pas tant que la totalité de l&#39;échantillon de grand champ, ce qui permet un plus grand rapport 23 signal-sur-bruit. En fonction de la résolution de la puissance du laser et le temps utilisé, les biomolécules peuvent être internalisés comptées (à l&#39;aide de l&#39;analyse pas à pas-photoblanchiment, figure 3), localisées, suivies ou 24-28. L&#39;internalisation des constructions doublement marquée avec une paire de fluorophores de FRET permet la quantification de FRET tant au unicellulaire ou niveaux seule molécule (figure 6). Différents paramètres peuvent être modifiésen fonction de la sortie souhaitée et le système biologique étudié. Tout d&#39;abord, la quantité de matière internalisé par la cellule peut être réglée en modifiant la concentration de biomolécules marquées ajouté aux cellules avant l&#39;électroporation (figure 2). l&#39;intensité de champ d&#39;électroporation aura aussi une influence à la fois l&#39;efficacité de chargement et la viabilité des cellules; comme prévu, tandis que l&#39;efficacité de chargement augmente avec l&#39;intensité du champ, la viabilité des cellules électroporées diminue (figure 4A). Les deux paramètres peuvent être quantifiés en enregistrant le pourcentage de charge et la division des cellules après l&#39;électroporation. Ce dosage de la viabilité couplée à l&#39;imagerie de fluorescence vérifie également l&#39;observation des biomolécules internalisés dans les cellules vivantes et permet une observation continue sur plusieurs générations (figure 4B). En résumé, ce protocole permet l&#39;internalisation des marquées par fluorescence des molécules ADN et de protéines dansE. coli ou S. 26 cerevisiae. Les molécules individuelles marquées avec des fluorophores organiques peuvent être suivis avec la résolution spatio-temporelle élevée pour des échelles de temps un ordre de grandeur plus de médecins de famille. Enfin, cette méthode est compatible avec grand champ, la FRBR et la détection confocale, ainsi que des systèmes d&#39;excitation pulsés, comme ALEX (alternant excitation laser 28,29).

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Name of Material/ Equipment</td>
			<td style="width:227px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:321px;">ElectroMax DH5-alpha Comptent cells</td>
			<td style="width:227px;">Invitrogen</td>
			<td style="width:119px;">11319-019</td>
			<td>or any other commercial or lab-mage electrocompetant bacteria or yeast.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">EZ Rich Defined Madia</td>
			<td style="width:227px;">Teknova</td>
			<td style="width:119px;">M2105</td>
			<td>low fluorescence rich media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroPulser Electroporation Apparatus&#160;</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:119px;">165-2100</td>
			<td>or any classical electroporator for microorganism transformation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Certified Molecular Biology agarose</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:119px;">161-3100</td>
			<td>low fluorescence agarose for agarose pad</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:321px;">Microscope coverslips No 1.5 thickness</td>
			<td style="width:227px;">Menzel</td>
			<td style="width:119px;">BB024060SC</td>
			<td>remove background particles by heating slides in furnace at 500 &#176;C for 1h</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Single-molecule fluorescence microscope</td>
			<td style="width:227px;">Home-built</td>
			<td style="width:119px;"></td>
			<td>described in REFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Localization software</td>
			<td style="width:227px;">Custom-written, available online</td>
			<td style="width:119px;"></td>
			<td>MATLAB and C++ software package that can be adapted for localization analysis.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Tracking software</td>
			<td style="width:227px;">Available online</td>
			<td style="width:119px;"></td>
			<td>MATLAB implementation by Blair and Dufresne.&#160;</td>
		</tr>
	</tbody>
</table>

internalization and observation of fluorescent biomolecules in living microorganisms via electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Watch this Scientific Journal Video about Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation at JoVE.com

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Internalisation et l&#39;Observation des Biomolécules Fluorescent In Living micro-organismes par électroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

internalization-observation-fluorescent-biomolecules-living

Research

JoVE Journal

Biology

16.7K vues.  University of Oxford. Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Vidéo: Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Electroporation de mycobactéries

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Recherche

Biologie

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imagerie de cellules vivantes de la F-actine fluorescente Dynamics via chatoiement de microscopie (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Culture primaire et l&#39;électroporation plasmidique de l&#39;Organe de Corti murin.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Changement de forme d&#39;imagerie cellulaire en vie Drosophile Embryons

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Suivi de l&#39;activité ubiquitine-protéasome dans les cellules vivantes aide d&#39;un Degron (dgn)-déstabilisé Green Fluorescent Protein Reporter Protéines (GFP) à base de

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

Protéines SAGESSE: un plan de travail pour<em&gt; In silico</em&gt;<em&gt; De novo</em&gt; Conception de biomolécules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

In Vivo L&#39;électroporation et microinjection de testicule de souris

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Suivi des modifications induites par la drogue dans des récepteurs post-internalisation traite par Analyse Colocalizational

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

Observation et Quantification de Mating Comportement dans le nématode,<em&gt; Bursaphelenchus xylophilus</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...