Die Studie wurde durch Zuschüsse der Fondazione Bambino Gesù und Ricerca Corrente (italienisches Gesundheitsministerium) an C.C.  Wir danken Dr. Enrico Bertini (Abteilung für Neurowissenschaften, Abteilung für neuromuskuläre und neurodegenerative Erkrankungen, Labor für Molekulare Medizin, Kinderforschungskrankenhaus Bambino Gesù), Dr. Stefania Petrini (Kerneinrichtung für Konfokale Mikroskopie, Forschungslabors, Kinderforschungskrankenhaus Bambino Gesù), Giulia Pericoli (Abteilung für Onkohämatologie, Gen- und Zelltherapie, Kinderforschungskrankenhaus Bambino Gesù) und Roberta Ferretti (Abteilung für Onkohämatologie, Gen- und Zelltherapie, Kinderforschungsklinik Bambino Gesù) für wissenschaftliche Diskussionen und technische Hilfe. Maria Vinci ist Empfängerin eines "Children with Cancer UK Fellowship".

1. Beschichtung von 96 Well-Platten
HINWEIS: Verschiedene Beschichtungen wurden in derselben Platte, aber separaten Vertiefungen getestet (siehe Zusatzdatei).
<ol><li>Verdünnen Sie das Matrigel 1:100 in DMEM. 100 μL pro Vertiefung in die 96 Wellplatten geben und 1 h bei Raumtemperatur inkubieren. Entfernen Sie anschließend die Lösung und waschen Sie die Vertiefungen zweimal mit 100 μL DMEM.</li>
	<li>Verdünnen Sie Laminin (20 μg/ml, LN-521) in PBS (mit Calcium und Magnesium). 100 μL in die Vertiefung geben und bei 4 °C über Nacht inkubieren. Am nächsten Tag führen Sie zwei Wäschen mit DMEM durch, bevor Sie die Zellen säen.</li>
	<li>Verdünntes Invitronektin (10 μg/ml) in Verdünnungspuffer. 100 μL pro Vertiefung in die 96-Well-Platte geben und 1 Stunde bei Raumtemperatur inkubieren. Waschen Sie die Vertiefungen mit PBS (ohne Kalzium und Magnesium), bevor Sie die Zellen plattieren.</li>
	<li>HU-Fibronektin (30 μg/ml) inddH2Overdünnen. 100 μL in die Vertiefungen geben und 45 Minuten bei Raumtemperatur inkubieren. Anschließend waschen Sie die Vertiefungen mit dem Medium, bevor Sie die Zellen säen.</li>
</ol>2. Aufrechterhaltung von iPSC in Kultur
HINWEIS: iPSCs wurden kommerziell erworben. Die iPS-Zellen wurden aus gesunden menschlichen Fibroblasten gewonnen und mittels episomaler Technologie umprogrammiert.
<ol><li>Aus dem -80 °C Gefrierschrank oder flüssigem Stickstoff die kryokonservierten iPSCs in einem 37 °C warmen Wasserbad auftauen. Reinigen Sie die Durchstechflasche mit den Zellen mit 70% Ethanol, bevor Sie sie in die biologische Sicherheitswerkbank bringen.</li>
	<li>Geben Sie die Zellsuspension in 5 mL vorgewärmtes Zellkulturmedium (z. B. mTeSR1) Tropfen für Tropfen mit einer 1000 μL Pipette in einem 15 ml sterilen konischen Röhrchen.</li>
	<li>Zentrifugieren Sie die Zellen bei 304 x g für 5 min bei Raumtemperatur (RT).</li>
	<li>Entfernen Sie das Medium und resuspendieren Sie das Zellpellet in 4 ml Zellkulturmedium.</li>
	<li>Die Zellsuspension wird in zwei Vertiefungen der 6 Wellplatten (105 iPSCs pro 6-Well-Zellkulturschale) gegeben, wo die embryonalen Fibroblasten (MEFs) der Maus zuvor plattiert wurden. Seed MEFs zwei Tage vor der Beschichtung von iPSCs mit einer Dichte von 2,4 x 104/cm2 in DMEM (enthält 10% Fetal Bovine Serum, 1% L-Glutamin und 1% Penicillin-Streptomycin).</li>
	<li>Nach der Aussaat ergänzen Sie die Zellmedien mit 10 μM ROCK-Inhibitor Y-27632.</li>
	<li>Züchten Sie die iPSCs auf MEFs für die ersten 4-5 Wochen und dann in dosierfreiem Zustand (MEF-freier Zustand), wobei eine der interessierenden Beschichtungen (siehe Schritt 1 und Tabelle 1) in mTeSR1 verwendet wird.</li>
	<li>Wenn die iPSCs zu 70-80% konfluent sind, Durchgang 1:4 mit 0,5 mM EDTA-Behandlung für 3-5 min bei RT. Fügen Sie 1 ml 0,5 mM EDTA für eine 6-Well-Platte (oder proportionale Mengen für andere Arten von Platten) hinzu. Überführung in neue Bohrlöcher unter feederfreien Bedingungen und Inkubation bei 37 °C, 5% CO2, 20%O2.</li>
	<li>Wechseln Sie die Medien täglich mit frischem mTeSR1 und teilen Sie die Zellen alle 2 Tage.</li>
</ol>3. Charakterisierung der Zellkonfluenz
<ol><li>Verwenden Sie 96 Well-Platten für die Experimente.</li>
	<li>Aussaat von 10.000 Zellen pro Vertiefung nach mindestens 1 Monat Kultivierung in feederfreiem Zustand, um sicher zu sein, dass die MEFs nicht passiert wurden. Verwenden Sie Einweg-Zählobjektträger, um die Zellen mit dem Lichtmikroskop zu zählen.</li>
	<li>Führen Sie die Experimente in dreifacher Ausführung durch. Testen Sie daher jede Aussaatbedingung in drei Vertiefungen.</li>
	<li>Führen Sie eine automatisierte Bildaufnahme ab Tag 1 nach der Aussaat mit einem Zytometer im Hellfeldmodus durch. Führen Sie eine automatisierte Bildaufnahme alle 24 Stunden für 5 Tage durch. Ausführliche Informationen zu den experimentellen Parametern finden Sie in der Zusatzdatei.</li>
	<li>Verwenden Sie Autokontrast und Autobelichtung, um Zellen besser sichtbar zu machen.</li>
	<li>Legen Sie die Analyseeinstellung (siehe Zusatzdatei) für die Konfluenzanalyse so fest, dass eine Maske von 60%, 80% und 100% pro Vertiefung angewendet wird, um die Fokusänderungen aufgrund der Lichtbrechung am Rand der Vertiefungen zu bewerten. Verwenden Sie die oben erwähnte andere Maskenanalyseeinstellung, um den Zellzusammenfluss zu jedem Zeitpunkt zu analysieren.</li>
</ol>4. Statistische Analysen
<ol><li>Geben Sie quantitative Ergebnisse als Mittelwert ± Standardfehler des Mittelwerts (SEM) an.</li>
	<li>Um die Gesamtunterschiede der verschiedenen Beschichtungsbedingungen zu vergleichen, erhalten Sie Daten mit denselben Proben und führen Sie den Paired-Sample-t-Test des Studenten durch. P-Werte kleiner als 0,05 gelten als statistisch signifikant, und alle gemeldeten p-Werte sind zweiseitig.</li>
</ol>5. Charakterisierung der Mikrofilamente des Zytoskeletts
<ol><li>Fixieren Sie Zellen mit 4% Paraformaldehyd (4% PFA) in PBS für 10 min bei RT, gefolgt von zwei Wäschen in PBS (10 min insgesamt).</li>
	<li>100 μL Blockierlösung (bestehend aus 5% BSA, 0,1% Triton in PBS) für 1 h RT in jede Vertiefung geben.</li>
	<li>Entfernen Sie die Blockierlösung und waschen Sie die Proben zweimal mit PBS für 10 min.</li>
	<li>100 μL der Phalloidin-konjugierten Arbeitslösung pro Probe zugeben und 1 h bei RT inkubieren.</li>
	<li>Waschen Sie die Zellen zweimal mit PBS (10 min bei RT).</li>
	<li>Färbekerne mit Hoechst 33342 verdünnt 1:10000 in PBS für 10 min bei RT.</li>
	<li>Entfernen Sie die Hoechst-Lösung und waschen Sie die Zellen zweimal mit PBS für jeweils 10 Minuten.</li>
	<li>Die Probe mitH2Owaschen und unter einer chemischen Haube trocknen lassen.</li>
	<li>Fügen Sie 100 μL Montagemedien (z. B. PBS: Glycerin, 1: 1) hinzu, um die Zellen abzudecken und die Fluoreszenz der Proben zu erhalten.</li>
	<li>Beobachten Sie die Zelle bei Ex/Em 493/517 nm auf einem konfokalen Laserscanning-Mikroskop, das mit einer Weißlichtlaserquelle (WLL) und einem 405-nm-Diodenlaser ausgestattet ist. Nehmen Sie sequenzielle konfokale Bilder mit einem HC PLAPO 40x Ölimmersionsobjektiv auf. Verwenden Sie die gleiche Laserleistung, Strahlteiler, Filtereinstellungen, Pinhole-Durchmesser und Scan-Modus für alle untersuchten Proben.</li>
</ol>

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Die Verwendung von iPS-Zellen für die Krankheitsmodellierung und das zukünftige Arzneimittelscreening zusammen mit ihrer möglichen Anwendung in der Präzisionsmedizin macht sie zu einer Technologie von großer Relevanz, und aus diesem Grund glauben wir, dass es notwendig ist, die In-vitro-Kultivierungsbedingungen klar zu verstehen, die der physiologischen Situation embryonaler Stammzellen besser ähneln. In diesem Zusammenhang haben wir verschiedene ECM-Beschichtungen mit Wildtyp-iPSCs getestet, um die Bedingungen zu ...

Induzierte pluripotente Stammzellen (iPS-Zellen) werden aus somatischen Zellen gewonnen und können in verschiedene Zelltypen differenziert werden. Sie werden oft als System zur Modellierung der Krankheitspathogenese oder zur Durchführung von Medikamentenscreenings eingesetzt und bieten auch das Potenzial, im Kontext der personalisierten Medizin eingesetzt zu werden. Da iPSCs ein großes Potenzial haben, ist es wichtig, sie für den Einsatz als zuverlässiges Modellsystem vollständig zu charakterisieren. Wir haben bereits gezeigt, wie wichtig es ist, iPS-Zellen in einer hypoxischen Umgebung zu züchten, da diese Zellen auf Glykolyse angewiesen sind und eine aerobe Umgebung ein Redoxungleichgewicht verursachen kann1. iPSCs sind auch anfällig für andere Kulturbedingungen, insbesondere die extrazelluläre Umgebung. Die Optimierung der Kulturbedingungen ist ein Schlüsselthema, um sie gesund zu halten und zu vermehren. Eine gesunde iPSC-Kultur führt zu gesunden, differenzierten Zellen, die im Allgemeinen der Endpunkt des Modells sind, das zum Verständnis molekularer, zellulärer und funktioneller Merkmale spezifischer menschlicher Störungen oder zellulärer Prozesse verwendet wird.
In dieser Studie wurde ein einfaches Protokoll verwendet, um den Zusammenfluss von iPSCs unter Verwendung verschiedener Beschichtungsbedingungen in separaten Vertiefungen zu testen. iPSCs benötigen eine Feederschicht von murinen embryonalen Fibroblasten (MEF), um richtig zu binden, aber die Koexistenz von iPSCs und MEF macht es schwierig, Analysen wie RNA- oder Proteinextraktion durchzuführen, da zwei Zellpopulationen vorhanden sind. Um die Feederschicht zu vermeiden, wurden verschiedene Proteine der extrazellulären Matrix (ECM) verwendet, um die natürliche Zellnische nachzubilden und eine feederfreie iPSC-Kultur zu haben. Insbesondere ist Matrigel ein lösliches Basalmembranpräparat, das aus dem Engelbreth-Holm-Swarm (EHS)-Maussarkom extrahiert wird und mit extrazellulären Matrixproteinen (d. h. Laminin, Kollagen IV, Heparansulfat-Proteoglykanen, Entactin/Nidogen und Wachstumsfaktoren) angereichert ist2,3. Die anderen verwendeten Beschichtungsbedingungen sind stattdessen gereinigte Proteine mit bekannter Relevanz für den Aufbau der ECMs: Laminin-521 ist dafür bekannt, von humanen pluripotenten Stammzellen (hPSCs) in der inneren Zellmasse des Embryos sezerniert zu werden, und es ist eines der häufigsten Lamininine im Körper nach der Geburt 4,5,6,7,8,9, 10,11; vitronectin ist eine xenofreie Zellkulturmatrix, von der bekannt ist, dass sie das Wachstum und die Differenzierung von hPSC 12,13,14,15,16 unterstützt; Fibronektin ist ein ECM-Protein, das für die Entwicklung von Wirbeltieren und die Anheftung und Erhaltung embryonaler Stammzellen in einem pluripotenten Zustand wichtig ist 17,18,19,20,21,22,23,24,25. Da unterschiedliche Beschichtungsbedingungen zur Verfügung stehen, vergleichen wir sie hinsichtlich ihrer Wirkung auf den Zusammenfluss von iPSCs.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
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		<col />
		<col />
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	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

Diese Studie konzentriert sich auf das Verständnis, wie wachsende iPS-Zellen auf verschiedenen ECM-Beschichtungssubstraten die Zellkonfluenz beeinflussen können. Es wurde ein Protokoll zur Bewertung der iPSC-Konfluenz in Echtzeit erstellt, ohne dass Zellen in Einzelzellsuspension gezählt werden müssen, um Wachstumsstörungen zu vermeiden. Ein High-Content-Bildanalysesystem wurde verwendet, um die iPCS-Konfluenz auf 4 verschiedenen ECMs im Laufe der Zeit automatisiert zu bewerten. Verschiedene Analyseeinstellungen wurden verwendet, um die Zellkonfluenz von adhärenten iPS-Zellen zu beurteilen, und es wurde nur ein geringer Unterschied (nach 24 und 48 Stunden mit Laminin) beobachtet, ob eine 60-, 80- oder 100%-Maske angewendet wurde. Wir zeigen auch, dass Laminin im Vergleich zu Matrigel, Vitronektin und Fibronektin zum besten Zusammenfluss führt.

In dieser Studie untersuchten wir den Zusammenfluss von iPSCs, wenn er unter verschiedenen Beschichtungsbedingungen gezüchtet wurde. Mit einem Zytometer konnten wir in 5 Tagen leicht aussagekräftige Ergebnisse in dreifacher Ausfertigung erhalten. Da iPSCs kaum an Kunststoffgefäßen haften und eine Beschichtung notwendig ist, um ihre Proliferation zu unterstützen, haben wir uns entschieden, den Zusammenfluss von humanen iPSCs zu überwachen, da dies auf die Gesundheit der Zellkultur hinweist und ihr Differenzierungspo...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

Das Ziel des Protokolls ist es, verschiedene extrazelluläre Matrix-Beschichtungsbedingungen (ECM) zu vergleichen, um zu beurteilen, wie sich eine differentielle Beschichtung auf die Wachstumsrate von induzierten pluripotenten Stammzellen (iPSCs) auswirkt. Insbesondere wollen wir Bedingungen schaffen, um ein optimales Wachstum von iPSC-Kulturen zu erreichen.

Messung des Zusammenflusses von iPSCs mit einem automatisierten Bildgebungssystem.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

measuring-the-confluence-of-ipscs-using-an-automated-imaging-system

Research

JoVE Journal

Biology

1.9K Aufrufe.  Ospedale Pediatrico Bambino Gesù, IRCCS. Das Ziel des Protokolls ist es, verschiedene extrazelluläre Matrix-Beschichtungsbedingungen (ECM) zu vergleichen, um zu beurteilen, wie sich eine differentielle Beschichtung auf die Wachstumsrate von induzierten pluripotenten Stammzellen (iPSCs) auswirkt. Insbesondere wollen wir Bedingungen schaffen, um ein optimales Wachstum von iPSC-Kulturen zu erreichen.

Serie: Measuring the Confluence of iPSCs Using an Automated Imaging System

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Bioelectric Analysen eines osseointegrierte Intelligent Implant Design System für Amputierte

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Forschung

Biologie

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Die Messung der Biegesteifigkeit von Bakterienzellen mit einer optischen Falle

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Eine einfache Methode zur Bildgebung Arabidopsis Leaves Mit Perfluordecalin als Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Messung der Auswirkungen von Bakterien auf<em&gt; C. Elegans</em&gt; Verhalten Mit einem Ei Retention Assay

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imaging Räumliche Reorganisation eines MAPK Signalweg Verwendung des Tabak Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Die Untersuchung der Proteinqualitätskontrolle System der<em&gt; D. discoideum</em&gt; Verwendung von Temperaturgesteuerte Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Ein schnelles automatisiertes Protokoll zur Muskelfaser Populationsanalyse in Rattenmuskelquerschnitte Mit Myosin Heavy Chain Immunhistochemie

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Flexible Messung der Biolumineszenz Reporter mit einer automatisierten längs Luciferase Imaging-Gas - und Temperatur-optimierte Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Errichtung einer extrazellulären sauren pH-Wert Kultursystem

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...