Die Autoren möchten die folgenden Finanzierungsquellen haben: Canadian Institutes of Health Research (CIHR), der Natural Sciences and Engineering Research Council (NSERC).

1. Entwicklung von LabVIEW-Programm <ol><li> Ein Massendurchflussregler (MKS M100B Masse-Flo-Controller) mit dem Heliumgasleitung angebracht, um eine präzise Gasflussrate zu gewährleisten. Diese Einheit wird von einer Eingangsspannung gesteuert wird, die zwischen 0 und 5 V. Eine Steuerschleife in der LabVIEW-Programm bestimmt die erforderliche Spannung zu einem bestimmten Zeitpunkt. Schnittstelle zwischen dem Computer und der Mengendurchflusssteuerung wird durch eine Datenerfassungseinrichtung (NI USB-6009) durchgeführt und eine 12-V-Stromversorgung versorgt den Massendurchflussregler. </li><li> Zwei Positionierungslinearführungen werden verwendet, um die Bewegung der Platte mit 6 Vertiefungen, einer für jede Richtung zu steuern. Jede Einheit besteht aus einer MA15-Serie Velmex Unislide Versammlung gekoppelt mit einem Schrittmotor und die beiden Baugruppen werden von einem einzigen Velmex VXM Schrittmotor-Controller gesteuert. Der Controller ermöglicht die manuelle Joggen jedes Motors oder akzeptiert eine Reihe von externen seriellen Befehle, deren Kombination ermöglicht gezielte Bewegung patterns. Mehrere Muster können in die LabView-Programm programmiert werden, dass der Benutzer nur die gewünschte Brennergeschwindigkeit sowie die Pfad Abmessungen liefern. </li></ol> 2. Vorbereitung der Ausrüstung <ol><li> Öffnen Sie sowohl die Helium-Flasche und Atemregler. </li><li> Schalten Sie den Motor-Controller. </li><li> Öffnen Sie das LabView Programm und alle Bedingungen zu überprüfen. </li><li> Achten Sie darauf, die Plattform zentriert ist. Um zu überprüfen, sehen sich an Motorstufe und sicherstellen, dass jeder Wagen ist entweder nicht in der Nähe der Bühne Ende. Entweder haben das Programm Mitte die Wagen oder manuell, indem Sie die Jogging-Bedienelemente auf der Vorderseite der Motorsteuerung anpassen. </li><li> Wenn dies der erste Lauf des Tages, ist es empfehlenswert, das Programm einmal ohne Zellen laufen, um sicherzustellen, das Setup richtig funktioniert. </li></ol> 3. Herstellung des Gasstrahls Kapillardüse Höhe <ol><li> Entfernen Sie die Kapillare aus der Halterung und die Höhe Null Zifferblatt. </li><li> Legen Sie die 6-Well-Platte on die Plattform. </li><li> Ersetzen und senken den Gasstrahl Kapillare auf den Grund des Brunnens, bis es das Deckglas trifft. Sichern Sie die Kapillare in die Halterung. </li><li> Über die Höhe Zifferblatt, erhöhen die Kapillare 3 mm. Markieren Sie diese Höhe auf der Kapillare Basis. </li><li> Heben Sie die Kapillare auf eine sichere Höhe und dann die 6-Well-Platte. </li></ol> 4. Zellpermeabilisierung Protokoll <ol><li> Kultur adhärenten Zelllinie, auf Glasabdeckung Konfluenz rutscht in einer 6-Well-Platte mit Standard-Kulturtechniken. Für HeLa-Zellen, Samen Platten mit 6 Vertiefungen mit 2 × 10 5 Zellen pro Vertiefung und Inkubation für 48 h vor der Behandlung. </li><li> Bereiten Sie entsprechende Volumen der Lösung, die das gewünschte Vektor. Für hrGFPII-1, eine Konzentration von 50 ng / ul in 1x PBS bietet eine starke Fluoreszenzsignal in HeLa-Zellen 24 Stunden nach der Transfektion. Für Dextran Studien wird 80 pg / ml 10 kDa grün fluoreszierenden Dextran empfohlen. Von nun an wird diese Lösung zu einer bezeichnets &quot;Vektor-Lösung.&quot; </li><li> Entfernen von Zellkulturmedien von gut und spülen dreimal mit 1x PBS. </li><li> Pipettieren 1370 ul Vektor Lösung in Schritt 4.2 in gut vorbereitet. Dies sollte in einer Flüssigkeitstiefe von ca. 1,3 mm führen. </li><li> Zeigen die auch den Vektor enthaltenden Lösung in der Mitte der Plattform. Senken Sie den Gasstrahl Düse auf die zuvor markierten Ebene einen Düsenhöhe von 3 mm über der Folie angezeigt wird. </li><li> Stellen Helium Strömungsgeschwindigkeit in der LabView-Programm und wählen Sie die gewünschte Bewegungsmuster verwendet werden. Wählen Sie &quot;Ausführen&quot;, wenn Sie bereit sind, um die Permeabilisierung Protokoll beginnen. Es wird eine anfängliche Verzögerungszeit, während der die Massenstrom-Steuer bereitet die notwendige Strömungsgeschwindigkeit bereitzustellen. Sobald die entsprechende Durchflussmenge erreicht ist, wird der LabView-Programm die Schrittmotoren zu aktivieren, die auch in der gewünschten Permeabilisierung Muster zu bewegen. Sobald die Plattform angehalten hat, warten Sie etwa 30 Sekunden und entfernen Sie den Vektor-Lösung.Hinweis: Optimale Permeabilisierung erfolgt an einem Staudruck von 100 bis 200 Pa am Düsenaustritt je nach Durchmesser der Kapillare. </li><li> Gut mit 1x PBS waschen dreimal. </li><li> Füllen gut mit frischem Kulturmedium. </li><li> Wiederholen Sie die Schritte von 4,3 bis 4,6 für die gewünschte Anzahl von Experimenten. Hinweis: Achten Sie darauf, Kontrollproben, die von Brunnen, die mit dem Vektor-Lösung ohne den Gasstrahl wird über sie laufen bestehen gefüllt sind. Die Lösung ist in den Kontrollvertiefungen für ca. 1 min dann zu den Schritten 4.5 und 4.6 gehen. </li><li> Arbeiten Sie mit einem hrGFP Transfektionsexperiment, kehren 6-Well-Platte für 24 Stunden Inkubator, damit transfizierten Material von den Zellen transkribiert werden. Wenn Sie mit einem Dextran Permeabilisierung Experiment zurück 6-Well-Platte für 15 min Inkubator. </li></ol> 5. Cell Imaging <ol><li> Wenn gewünscht, unmittelbar vor der Bildgebung, Kontrastfärbung mit entsprechenden Live / Dead-Färbung, um den Zelltod zu evaluieren. </li><li> Bild Zellen mit angemesriate Mikroskop der Transfektion / Permeabilisierung Wirksamkeit zu untersuchen. </li></ol>

<ol>
	<li>Rosenberg, S. 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=Gene+transfer+into+humans--immunotherapy+of+patients+with+advanced+melanoma,+using+tumor-infiltrating+lymphocytes+modified+by+retroviral+gene+transduction.">Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.</a> The New England Journal of Medicine. 323, 570-578 (1990).</li><li>Yang, 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=Cellular+immunity+to+viral+antigens+limits+E1-deleted+adenoviruses+for+gene+therapy.">Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 4407-4411 (1994).</li><li>Chen, C., Okayama, H. <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+mammalian+cells+by+plasmid+DNA.">High-efficiency transformation of mammalian cells by plasmid DNA.</a> Molecular and cellular biology. 7, 2745-2752 (1987).</li><li>Walev, I., 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=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 3185-3190 (2001).</li><li>Felgner, P. 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=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 7413-7417 (1987).</li><li>Hamm, A., Krott, N., Breibach, I., Blindt, R., Bosserhoff, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+method+for+primary+cells.">Efficient transfection method for primary cells.</a> Tissue engineering. 8, 235-245 (2002).</li><li>Rauth, S., Kucherlapati, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+DNA+Transferred+into+Mammalian-Cells.">Expression of DNA Transferred into Mammalian-Cells.</a> J. Bioscience. 6, 543-567 (1984).</li><li>Spandidos, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-mediated+gene+transfer+(electroporation)+into+mouse+Friend+and+human+K562+erythroleukemic+cells.">Electric field-mediated gene transfer (electroporation) into mouse Friend and human K562 erythroleukemic cells.</a> Gene analysis techniques. 4, 50-56 (1987).</li><li>Chu, G., Hayakawa, H., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+for+the+efficient+transfection+of+mammalian+cells+with+DNA.">Electroporation for the efficient transfection of mammalian cells with DNA.</a> Nucleic Acids Research. 15, 1311-1326 (1987).</li><li>Fechheimer, 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=Transfection+of+mammalian+cells+with+plasmid+DNA+by+scrape+loading+and+sonication+loading.">Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 8463-8467 (1987).</li><li>Koch, S., Pohl, P., Cobet, U., Rainov, 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=Ultrasound+enhancement+of+liposome-mediated+cell+transfection+is+caused+by+cavitation+effects.">Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects.</a> Ultrasound in Medicine &amp; Biology. 26, 897-903 (2000).</li><li>Greenleaf, W. J., Bolander, M. E., Sarkar, G., Goldring, M. B., Greenleaf, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+cavitation+nuclei+significantly+enhance+acoustically+induced+cell+transfection.">Artificial cavitation nuclei significantly enhance acoustically induced cell transfection.</a> Ultrasound in Medicine &amp; Biology. 24, 587-595 (1998).</li><li>Yang, N. S., Burkholder, J., Roberts, B., Martinell, B., McCabe, D. <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+and+in+vitro+gene+transfer+to+mammalian+somatic+cells+by+particle+bombardment.">In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.</a> Proceedings of the National Academy of Sciences of the United States of America. 87, 9568-9572 (1990).</li><li>Hallow, D. 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=Shear-induced+intracellular+loading+of+cells+with+molecules+by+controlled+microfluidics.">Shear-induced intracellular loading of cells with molecules by controlled microfluidics.</a> Biotechnology and Bioengineering. 99, 846-854 (2008).</li><li>Chouinard-Pelletier, G. L., Guay, M., Coulombe, D., Leask, S., L, R., Jones, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+inert+gas+jets+to+measure+the+forces+required+for+mechanical+gene+transfection.">Use of inert gas jets to measure the forces required for mechanical gene transfection.</a> Biomedical Engineering Online. 11, (2012).</li><li>Leduc, M., Coulombe, S., Leask, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Plasma+Jet+Deposition+of+Patterned+Polymer+Films+for+Cell+Culture+Applications.">Atmospheric Pressure Plasma Jet Deposition of Patterned Polymer Films for Cell Culture Applications.</a> Ieee T. Plasma Sci. 37, 927-933 (2009).</li><li>Lu, 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=Microfluidic+shear+devices+for+quantitative+analysis+of+cell+adhesion.">Microfluidic shear devices for quantitative analysis of cell adhesion.</a> Analytical Chemistry. 76, 5257-5264 (2004).</li><li>Sankin, G. N., Yuan, F., Zhong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsating+tandem+microbubble+for+localized+and+directional+single-cell+membrane+poration.">Pulsating tandem microbubble for localized and directional single-cell membrane poration.</a> Physical Review Letters. 105, 078101 (2010).</li></ol>

Die Autoren erklären, sie haben keine finanziellen Interessen konkurrieren.

Schutzgasstrahl Permeabilisierung ist eine nützliche Technik für adhärenten Zell Transfektion. Es bietet die Möglichkeit, Biomoleküle in Zellen unter Verwendung von mechanischen Kräften, die die Notwendigkeit einer potentiell schädlichen Chemikalien oder virale Vektoren beseitigt übertragen. Die Technik könnte möglicherweise geben Forscher und Kliniker eine effiziente und relativ einfache Möglichkeit, genau Transfektion von Zellen. Die Selektivität der Permeabilisierung ist auch einzigartig so dass die Forsc...

Mit der Entwicklung der Biomedizin und das Verständnis der Zellmechanik, die Lieferung von Biomolekülen in Zellen ist unerlässlich geworden, um viele Forschungsfelder und medizinische Therapien. Verschiedene Techniken wurden entwickelt, um Fremdmolekülen durch die Plasmamembran und in das Zell-Cytosol einzuführen. Virale, chemische oder mechanische Techniken: Diese können in der Regel entweder als klassifiziert werden. Viral Techniken genetisches Material wie DNA und RNA mittels viraler Vektoren 1 transfizieren. Viral-Techniken sind in der Regel hohe Wirkungsgrade in bestimmten Zelllinien haben, aber sie haben das Potenzial für Immun-und Entzündungsreaktionen 2. Allgemeine chemische Transfektion Methoden gehören Fällung mit Calciumphosphat 3, bakterielle Exotoxine 4 und 5 Lipofektion. Diese Techniken haben sich als wirksam erwiesen, jedoch ergeben sich gewisse Probleme wie Zelltoxizität und Nicht-Spezifität. Weiterhin Techniken wie Calciumphosphate Niederschlag gefunden Transfektion Wirkungsgrade bis zu 70%, aber nur in bestimmten Zelllinien, primären Zellen selten in 6. Dies ist bemerkenswert, da zunehmende Forschungsanstrengungen werden in primären Zellen, insbesondere bei der Gestaltung verschiedene Behandlungen für die klinische Anwendung und die Untersuchung der DNA-Funktion setzen. Zeitliche Unterbrechung der Zellmembran durch mechanische Reize ist eine Alternative zur Einführung von Fremdmolekülen in die Zellen. Techniken umfassen: Mikroinjektion von Molekülen 7, Elektroporation unter Verwendung eines elektrischen Feldes, um die Membran 8,9 stören Sonoporation mit Ultraschallwellen, um die Zellmembran 10-12, Partikelbeschuss stören, wie durch das &quot;Gen-Kanone&quot;, die Teilchen gebunden schießt nachgewiesen Gene in der Zelle 13, und in jüngster Zeit die Anwendung von Schubspannung, die gezeigt worden ist, zeitlich permeabilisiert Säugerzellen 14,15. Obwohl Mechanikeral Methoden vermeiden einige der oben genannten Probleme, werden sie in der Regel durch geringere Wirkungsgrade und sehr komplexen und spezialisierten Setups begleitet. Ein Atmosphärendruck-Glimmentladung Brenner (APGD-t) wurde an der McGill mit dem anfänglichen Ziel der Funktionalisierung von Oberflächen-und Abnahme adhärenten Zellen in vitro 16 entwickelt. Serendipitously wurde entdeckt, dass eine lebend / tot-Färbung verwendet wurde, irgendwie in die von Zellen ohne Permeabilisierung Mittel versetzt entnommen. Außerdem schien dies nur in eingeschränkten Bereichen der Vertiefungen, die mit der Fackel Pfad übereinstimmen passiert aufgetreten. Untersuchung der Permeabilisierung Fähigkeiten des APGD-t fortgesetzt und in Kontrollstudien wurde festgestellt, daß die Träger inerten Gasstrahl des Plasma ohne Anregung war auch in der Lage, Zellen zu permeabilisieren. Dies wider die anfängliche Hypothese, dass die durch den Plasmastrahl reaktiven Spezies vorübergehend das Membranfunktion beeinträchtigt und schlug vor, einfach die mechanischeschen Kräfte waren ausreichend, um Zellpermeabilisierung verursachen. Von hier, in unserem Labor weiter Studien zu versuchen und zu quantifizieren und charakterisieren die Permeabilisierung Effizienz eines inerten Gasstrahl sowie Blick auf seine Fähigkeiten Transfektion 16. Durch diese Untersuchungen wurde gefunden, dass Mikroporen in der Tat in Form der Plasmamembran, und diese Poren neigen dazu, innerhalb von etwa 5 Sekunden 15 verschließen. Wir haben Nützlichkeit der Technik der in vitro und in vivo in der Chorioallantoismembran eines Hühnerembryos zeigten.

<table border="1">
	<tbody>
		<tr>
			<td>Vitality hrGFPII-1</td>
			<td>Agilent Technologies Canada</td>
			<td>240143-57</td>
			<td>Green fluorescent protein for transfection experiments on HeLa cells</td>
		</tr>
		<tr>
			<td>Dextran, AlexaFluor 488; 10,000 MW, Anionic, Fixable</td>
			<td>Life Technologies Inc.</td>
			<td>D22910</td>
			<td>dextran for permeabilization experiments</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Life Technologies Inc.</td>
			<td>L3224</td>
			<td>for counterstaining of mammalian cells</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>for mounting slides when imaging</td>
		</tr>
		<tr>
			<td>Ultra High Purity Helium</td>
			<td>Praxair Canada Inc.</td>
			<td>HE 5.0UH-T</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone DMEM/ High Glucose Media - 500 ml</td>
			<td>Thermo Scientific</td>
			<td>SH30022.01</td>
			<td>for HeLa cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) 1x</td>
			<td></td>
			<td></td>
			<td>Produced in lab</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. List of required reagents</td>
		</tr>
		<tr>
			<td>6-Well Clear TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>#1 22 x 22 Coverslips</td>
			<td>Fisher</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td>WPI Sizing Information</td>
		</tr>
		<tr>
			<td>ID (mm): 1, 0.86, 0.68, 0.5 Order #: 1B200, 1B150, 1B120, 1B100</td>
		</tr>
		<tr>
			<td>Mass-Flo Controller</td>
			<td>MKS</td>
			<td>M100B01314CS1BV</td>
			<td>Mass flow controller, Series M100B, requires an external 12 V power supply</td>
		</tr>
		<tr>
			<td>USB-6009 DAQ Device</td>
			<td>National Instruments</td>
			<td>779026-01</td>
			<td></td>
		</tr>
		<tr>
			<td>UniSlide Assembly with stepping motor and controller</td>
			<td>Velmex</td>
			<td></td>
			<td>Two series MA15 UniSlide Assemblies, fitted with Vexta 1.8&#176; stepping motors provided by Velmex, and controlled by a VXM Stepping Motor Controller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. List of required equipment</td>
		</tr>
	</tbody>
</table>

permeabilization of adhered cells using an inert gas jet

Verschiedene Zell Transfektion Techniken existieren und diese können bis zu drei Kategorien unterteilt werden: Virus-, chemische und mechanische. Dieses Protokoll beschreibt ein mechanisches Verfahren zur zeitlich permeabilisiert adhärenten Zellen unter Verwendung eines inerten Gasstrahl, der die Übertragung von in der Regel nicht durchlässig Makromolekülen in Zellen erleichtern können. Wir glauben, dass diese Technik durch Verleihen von Scherkräften an der Plasmamembran von adhärenten Zellen, was die temporäre Bildung von Mikroporen. Wenn diese Poren erzeugt werden, werden die Zellen dann durchlässig genetische Material und anderen Biomolekülen. Die beteiligten mechanischen Kräfte nicht Gefahr laufen, dauerhaft beschädigen oder Ablösen Zellen von ihrem Substrat. Es ist daher ein enger Bereich des inerten Gasdynamik in denen die Technik effektiv ist. Eine inerte Gasstrahl hat bei permeabilisierenden verschiedenen adhärenten Zelllinien, einschließlich HeLa, HEK293 und menschlichen Bauchaorta-Endothelzellen effizient erwiesen. Dieses Protokoll für die p angemessenermeabilization von adhärenten Zellen in vitro und, wie wir gezeigt haben, in vivo, welche es für die Forschung und möglicherweise in zukünftige klinische Anwendungen verwendet werden. Es hat auch den Vorteil, permeabilisierenden Zellen in einem räumlich restriktiv, was beweisen könnte, um ein wertvolles Forschungsinstrument sein.

Permeabilisierung von HeLa-Zellen mit 10 kDa grün fluoreszierenden Dextran mit einem Heliumgasstrahl mit einem Innendurchmesser von 0,86 mm Kapillare ist in Fig. 1 gezeigt. Die Zellen wurden unmittelbar nach Permeabilisierung mit 2 ul / ml EthD-1-Lösung (LIVE / DEAD Lebensfähigkeit / Zytotoxizität Kit), um den Zelltod zu visualisieren gegengefärbt. Unmittelbar nach Gegenfärbung wurden die Deckgläser angebracht und abgebildet. Diese Figur zeigt Ergebnisse der Ausführung des Heliumgasstrahl auf dr...

Watch this Scientific Journal Video about Permeabilization of Adhered Cells Using an Inert Gas Jet at JoVE.com

Permeabilization of Adhered Cells Using an Inert Gas Jet

Dieses Protokoll beschreibt ein Verfahren für die vorübergehende Permeabilisierung der adhärenten Zellen unter Verwendung eines inerten Gasstrahl. Diese Technik erleichtert die Übertragung von genetischem Material und Biomolekülen in adhärente Säugetierzellen durch die Verwendung von mechanischen Kräften, um die Plasmamembran zu stören.

Permeabilisierung der anhaftenden Zellen mit einem Inertgas Jet

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes ...

permeabilization-of-adhered-cells-using-an-inert-gas-jet

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9.6K Aufrufe.  McGill University.  Dieses Protokoll beschreibt ein Verfahren für die vorübergehende Permeabilisierung der adhärenten Zellen unter Verwendung eines inerten Gasstrahl. Diese Technik erleichtert die Übertragung von genetischem Material und Biomolekülen in adhärente Säugetierzellen durch die Verwendung von mechanischen Kräften, um die Plasmamembran zu stören. 

Serie: Permeabilization of Adhered Cells Using an Inert Gas Jet

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Qualitative Charakterisierung der wässrigen Fraktion von hydrothermale Verflüssigung von Algen mit Hilfe von 2D-Gaschromatographie mit Time-of-Flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Forschung

Biotechnik

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

Quantifizierung von Mikroorganismen in geringen Konzentrationen mit Hilfe digitalen holographischen Mikroskopie (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

Hochauflösende Musterung mit zwei Modi der Electrohydrodynamic Jet: Drop-on-Demand und Nahfeld-Elektrospinnen

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Eine neuartige Operationstechnik als Grundlage für In Vivo teilweise Leber Engineering in der Ratte

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Reinigung und Analyse eines monoklonalen Antikörpers aus chinesischen Hamster-Ovarialzellen mit einem automatisierten Mikrobioreaktorsystem

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

Screening und Identifizierung von kleinen Peptiden Targeting Fibroblast Growth Factor Receptor2 mit einem Phage Display Peptid Bibliothek

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Herstellung und Betrieb eines kontinuierlichen Durchfluss-Mikroelektroporationssystems mit Permeabilisierungsdetektion

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

Formulierung und Charakterisierung eines Exosom-basierten Dopaminträgersystems

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...

Synthese von Lipid-Nanopartikeln mit CIJ- und μMIVM-Mischtechnologien

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.

Autor im Rampenlicht: Verbesserung der Bildung von Lipid-Nanopartikeln durch turbulentes Mischen in begrenzten Geometrien

A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (&#181;MIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

Synthese von Lipid-Nanopartikeln durch turbulente Strömung in geschlossenen Aufprallstrahlmischern

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of ...