Diese Studie wurde vom NIH Forschungsförderung (DK090868 und GM092930 zu TI) unterstützt. Es gibt eine schwebende Patentanmeldungen auf einem Käfig Rapamycinanalog.

1. Die Transfektion von Plasmid-DNA <ol><li> In Plasmid-DNA, 0,5 ug Membran-gebundenen FRB (nachstehend bezeichnet LDR) und 0,5 ug FKBP-TIAM1 (T-Zell-Lymphom Invasion und Metastasierung-induzierenden Proteins 1: Rac Aktivator) mit fluoreszierendem Protein auf 37,5 ul dH 2 O markiert Fügen Sie 1 ul FuGENE HD. </li><li> Vortex und Inkubation bei Raumtemperatur für 20 Minuten. Inzwischen, um Schritte von 1,3 bis 1,8 gehen. </li><li> Waschen Sie jede Vertiefung eines 8-Well-Kammer mit 50 ul Poly-D-Lysin. </li><li> Waschen der Mitte jeder Vertiefung mit dH 2 O </li><li> 80-85% konfluent trypsinieren NIH-3T3-Zellen und mit Wasser auf 10 ml Kulturmedium (DMEM mit 10% FBS). </li><li> Nehmen Sie 375 ul von Federungs-und zentrifugieren bei 1750 rpm für 3 Minuten. </li><li> Saugen Sie die Medien, die man aufpassen, nicht, um das Pellet zu stören. </li><li> Fügen Sie 750 ul DMEM w / 10% FBS und resuspendieren. </li><li> Fügen Sie Zellsuspension dH 2 O / DNA / FuGENE HD-Lösung und vorsichtig mischen. </li><li> Eintragen von 250ul Zell / DNA-Suspension in jede Vertiefung (füllt bis zu 3 Brunnen). </li><li> Inkubieren der transfizierten Zellen bei 37 ° C und 5% CO 2 für 15-18 Stunden. </li></ol> 2. Vorbereitung der CRB-A-Lösung <ol><li> Synthetisieren Käfig Rapamycin-Biotin-Addukt (CRB). Siehe &quot;Synthetische Schema der CRB&quot; für Details. Kurz gesagt werden Käfighaltung und Biotinreste separat hergestellt. Die Einheit wird dann Einsetzens konjugiert Rapamycin (LC lab), das Produkt anschließend an das Biotin-Komponente durch eine mittels Klickreaktion 7 bis CRb leisten gekoppelt ist. </li><li> Bereiten Sie eine 1 uM Stammlösung der CRB in DMSO. </li><li> Auflösen 1mg Avidin in 250 ul PBS in einem 0,5 ml Eppendorf-Röhrchen. </li><li> Fügen Sie 1 ul der CRB-Stammlösung zu Avidin aufgelöst. Mischen Sie durch mehrmaliges Wenden. </li><li> Bei Raumtemperatur für 15 min auf CRB-Avidin-Konjugat (CRB-A liefern)-Lösung. </li><li> Ausschließen ungebundenen kleine Moleküle und reinigen CRB-A mit einem Ausschluss-Säule. </li></ol> 3. Microscopy and Light Beleuchtung an subzellulären Ebene <ol><li> Serum hungern transfizierten Zellen in DMEM ohne FBS für 12 Stunden. </li><li> Vorsichtig absaugen Sie die Medien aus den Zellen und 250 ul der vorbereiteten 400 nM CRB-Eine Lösung (aus Schritt 2.6). </li><li> Zur Dynamik von Proteinen in lebenden Zellen an subzelluläre Auflösung vor und nach der Behandlung Licht sichtbar zu machen, wurde Spinning-Disk-Mikroskopie (Abbildung 2a). UV-Beleuchtung wurde durch die Kopplung Optiken für UV-LED-Licht (365 nm) mit Auflicht-Fluoreszenz Licht (Abbildung 2b) erreicht. </li><li> MetaMorph Software wurde benutzt, um Fluoreszenz-Aufnahmen von transfizierten Zellen alle 15 Sekunden zu erfassen. </li><li> Definieren der Koordinaten und die Größe eines Beleuchtungsfleck mit einer Glasplatte mit fluoreszierenden Farbstoffen, die durch UV-Licht angeregt beschichtet. Die Größe kanndurch die Anwendung Objektiv mit unterschiedlichen Vergrößerungen und durch die Auswahl optischer Fasern mit verschiedenen Core-Größe variiert werden. </li><li> Nach der Bestimmung Hintergrundwerte der Membran Kräuseln, UV-Licht bestrahlt an der Peripherie der Zellen. Die folgenden beiden Schritte sind, um den Kontrast vor lokalisierte Effekte. </li><li> Weltweit bestrahlen Zellen mit 365 nm Licht einer Quecksilberlampe bereitgestellt. </li><li> Lösen Sie demaskiert, so original Rapamycin in DMSO und dann in PBS (400 nm endg.), und fügen Sie die Lösung für das Imaging-Kammer zur globalen Effekte zu induzieren. </li></ol> 4. Repräsentative Ergebnisse Ein Beispiel für repräsentative Ergebnisse ist in 3 gezeigt. UV-Licht-Bestrahlung mit einer kleinen Bereich in der Nähe einer Kante zellulären schnell Bildung von lokalisierten induzierte Rüschen nur in und um den bestrahlten Bereich. Um zu bestätigen, dass dies tatsächlich lokale Aktivität, wir dann global Zellen mit UV bestrahlt, oder hinzugefügt Rapamycin zu den Bulk-Medien, die beide which induzierte globale Rüsche Bildung in der gesamten Membran. Für eine quantitative Analyse, teilten wir Zielzellen in vier Quadranten, und zählte die Häufigkeit der Rüsche Bildung in jedem Quadranten auf lokale als auch globale nach Stimulation unter verschiedenen Bedingungen einschließlich der Kontrollen (Abbildung 4). Es ist nur diese Kombination von UV-Bestrahlung, CRB-A, und geeignete FKBP-FRB-Konstrukten, die lokalen Kräuseln induziert. <img alt="1" src="/files/ftp_upload/3794/3794fig1.jpg" /> Abbildung 1. Schematische Darstellung der räumlich begrenzten Proteindimerisierung mit Käfig Rapamycin-Avidin-Konjugat (CRB-A) und UV-Licht. UV-Bestrahlung spaltet die Linker zwischen Rapamycin und Biotin, was zur Freisetzung von chemischen Dimerisierer (HE-Rapa, C40-O-(2-Hydroxyethyl)-rapamycin) und dem Nebenprodukt. Das freigesetzte Dimerisierer diffundiert dann in die Zellen und induziert die Dimerisierung zwischen FKBP-POI (Protein von Interesse) und Plasma membrane verankert FRB nur in der Nähe des bestrahlten Bereichs (blauer Kreis). Ohne UV-Bestrahlung, tun FKBP und FRB nicht Associate (rotes Kreuz), es kommt zu keinen Effekt. (Copyright @ ACS2011) 6 <img alt="2" src="/files/ftp_upload/3794/3794fig2.jpg" /> Abbildung 2. Bild des angepassten konfokalen Fluoreszenzmikroskop. (A) Frontalansicht des konfokalen Mikroskop (Axiovert 200, Zeiss). (B) Nahaufnahme, Draufsicht einer Koppeleinheit für EPI-Fluoreszenz-und UV-Beleuchtung. <img alt="Abbildung 3" src="/files/ftp_upload/3794/3794fig3.jpg" /> Abbildung 3. Räumlich begrenzten Rüsche Bildung in einer transfizierten NIH3T3-Zellen durch lokalisierte UV-Bestrahlung induziert. Konfokale Fluoreszenzbilder einer NIH3T3-Fibroblasten, die mit YFP-markierten FKBP-TIAM1 (Rac-Aktivator) und LDR (FRB-Membran verankert) transfiziert wird unterzog Bestrahlung in einem begrenzten Gebiet in der Nähe ihres Randes mit LED Licht bei 365 nm für 15 Sekunden ab 210 Sek. in Gegenwart von CRB-A-Konjugat in den Bulk-Medien. Diese Behandlung wurde von Global Illumination mit Quecksilber-Lampe für 1 s bei 1063 Sek. und globale Zugabe von Rapamycin letzten 400 Nm bei 1.560 Sek. gefolgt. Die Zelle zeigte fast keine zerzauste Aktivitäten zu Beginn (a), zeigte aber räumlich begrenzten Kräuseln nach lokalisierten Beleuchtung (gelber Kreis) (b). Die Zelle zeigte globalen Rüsche Bildung nach Global Illumination und Zugabe von Rapamycin (CD), was darauf hinweist, dass die lokale Bildung von Rüsche räumlich begrenzten Aktivierung von Rac verursacht wurde. Maßstab: 10 um. (Copyright @ ACS2011) 6 <img alt="Abbildung 4" src="/files/ftp_upload/3794/3794fig4.jpg" /> Abbildung 4. Häufigkeit der Rüsche Bildung in den Quadranten von transfizierten NIH3T3-Zellen. Lokale UV-Bestrahlung mit CRB-A und UV-Bestrahlung folgende globale wurden am 13. Zellen durchgeführt. CRB-A </sTrong&gt; Addition (keine UV-Bestrahlung), wurden lokale UV-Bestrahlung mit oder ohne Avidin auf 13, 5 und 6 Zellen durchgeführt, jeweils. Keine der untersuchten Zellen zeigten Rüschen Formation mit CRB-A ohne UV-Bestrahlung oder lokale UV-Bestrahlung mit oder ohne Avidin den ersten Medien. Wir haben Daten aus den Zellen mit geringen Hintergrund Rüschen Bildung, aber die in der Lage waren von Rapamycin-induzierte Bildung Rüschen, die wir nach jedem Experiment überprüft durch Bestätigen globalen Rüschen Bildung als Folge der Zugabe von Rapamycin (finalen 400 nM). Diese Ergebnisse zeigten, dass CRB-A und UV-Bestrahlung lokalen Ort brauchen Rüschen-Bildung in den Zellen sind. (Copyright @ ACS2011) 6 Abkürzungen: FKBP: FK506-bindende Protein FRB: FKBP-Rapamycin-bindenden Proteins TIAM1: T-Zell-Lymphom Invasion und Metastasierung-induzierenden Protein 1 (Rac-GEF) LDR: Lyn-Linker DSAG-FRB (Lyn: N-terminalen 11 Aminosäuren von Lyn-Kinase) HE-Rapa: C40-O - (2-Hydroxyethyl)-rapamycin CRB: Käfig mit Rapamycin Biotinanteil CRB-A: CRB konjugiert an Avidin

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Keine Interessenskonflikte erklärt.

Wir beschrieben eine Technik, die eine neuartige Verbindung Käfigen beschäftigt zusammen mit dem FKBP-FRB Heterodimerisierung System, um Rho GTPase-Aktivität zu einem bestimmten subzellulären Ort auf einer Zeitskala von Sekunden zu manipulieren. Es gibt drei Einschränkungen des vorliegenden Ansatzes. Erstens, weil das Verfahren zur Verhinderung oder die Diffusion von Dimerisierer über die Plasmamembran basiert, muss das Ziel zelluläre Lokalisation die Plasmamembran oder deren Umgebung...

<table border="1"><tbody><tr><td> Name des Reagenz / Ausrüstung </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Rapamycin </td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE HD </td><td> Roche </td><td> 04709691001 </td></tr><tr><td> Avidin </td><td> Sigma </td><td> A9275-10MG </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650-5X5ML </td></tr><tr><td> Größenausschlußsäule </td><td> GE Healthcare </td><td> Spin-Trap-G-25 </td></tr><tr><td> Glasboden 8-Well-Kammer </td><td> Thermo Scientific </td><td> 12-565-47 </td></tr><tr><td> Invertierten Fluoreszenzmikroskop </td><td> Zeiss </td><td> Axiovert200M </td></tr><tr><td> UV-LED-Lichtquelle </td><td> Rapp Opto Electronics </td><td> UVILED </td></tr><tr><td> Konfokale drehende Scheibe </td><td> Yokogawa Electric Corp </td><td> CSU10 </td></tr><tr><td> CCD-Kamera </td><td> Hamamatsu Photonics </td><td> ORCA-ER </td></tr><tr><td> 100x Objektiv </td><td> Zeiss </td><td> Plan Apochromat </td></tr></tbody></table> Syntheseschema der CRB (Copyright ACS2011) 6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="Schema 1" /></a> Synthetische Bedingungen: (a) BnBr, K 2 CO 3, DMF (b) f.HNO 3, AcOH (c) TFA (d) Propargyl-Br, K 2 CO 3, DMF, (e) Glycerin, Katze. PTSA, Toluol (f) NaBH 3 CN, TiCl 4, MeCN (g) Tf 2 O, Pyridin (h) Methanol-HCl, (i) LiAlH 4, THF (j) TsCl, Pyridin, CH 2 Cl 2 (k) NaN 3, DMF (l) 8, 2,6-Di-t-Butylpyridin, CH 2 Cl 2 (m) 13, CuSO 4, Ascorbat, 2-Propanol, H 2 O, CH 2 Cl 2.

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

Dynamischen Regulation der Rho-Familie kleiner Guanosin Triphosphatasen (GTPasen) mit großer Präzision raumzeitlichen ist essentiell für verschiedene zelluläre Funktionen und Ereignisse 1, 2. Ihre raumzeitlich dynamische Natur wurde durch Visualisierung der ihre Aktivität und Lokalisierung in Echtzeit 3 enthüllt worden. Um ein tieferes Verständnis ihrer Rollen in diversen zellulären Funktionen auf molekularer Ebene zu gewinnen, sollte der nächste Schritt Störung der Protein-Aktivitäten sein, an einer präzisen subzelluläre Ort und Zeitpunkt. (1) Rapamycin-FKBP-induzierte FRB Heterodimerisierung und (2) ein Foto einschließende Verfahren Rapamycin: Um dieses Ziel zu erreichen, haben wir ein Verfahren zur lichtinduzierten, räumlich-zeitlich kontrollierte Aktivierung der kleinen GTPasen durch Kombinieren von zwei Techniken entwickelt. Mit der Verwendung von Rapamycin-FKBP-vermittelte Heterodimerisierung FRB, haben wir ein Verfahren zur schnellen induzierbare Aktivierung oder Inaktivierung der kleinen GTPasen includi entwickeltng Rac 4, Cdc42 4, RhoA 4 und Ras 5, bei dem Rapamycin induziert Translokation von FKBP-kondensierten GTPasen, oder deren Aktivatoren, der Plasmamembran, wo FRB verankert ist. Für die Kopplung mit diesem Heterodimerisierung System haben wir auch ein Foto-Käfighaltung von Rapamycin-Analoga-System entwickelt. Ein Foto-caged Verbindung ist ein kleines Molekül, dessen Aktivität mit einem photospaltbaren Schutzgruppe als Käfighaltung Gruppe bekannt unterdrückt. Um Heterodimerisierung Aktivität vollständig zu unterdrücken, haben wir ein Käfig Rapamycin, die an ein Makromolekül, so dass die daraus resultierende große Komplex kann nicht über die Plasmamembran gebunden wird, was zu praktisch ohne Hintergrund-Aktivität als eine chemische Dimerisierer innerhalb der Zellen 6. Abbildung 1 zeigt ein Schema unserer System. Mit der Kombination dieser beiden Systeme haben wir vor Ort rekrutierten eine RAC-Aktivator zur Plasmamembran auf einer Zeitskala von Sekunden und erreicht durch Licht induzierten Rac-Aktivierung an der subzellulären levEL 6.

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Verfahren zum räumlichen-zeitlichen Steuerung der GTPase-Aktivität durch Licht beschrieben. Dieses Verfahren basiert auf Rapamycin-FKBP-induzierte FRB Heterodimerisierung und Foto einschließende Systeme. Optimierung der Licht-Bestrahlung ermöglicht die raum-zeitlich kontrollierte Aktivierung der kleinen GTPasen auf subzellulärer Ebene.

Räumlich-zeitliche Manipulation kleiner GTPase-Aktivität in subzellulären Ebene und auf Zeitskala von Sekunden in lebenden Zellen

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

spatio-temporal-manipulation-small-gtpase-activity-at-subcellular

Research

JoVE Journal

Bioengineering

10.7K Aufrufe.  Johns Hopkins University. Verfahren zum räumlichen-zeitlichen Steuerung der GTPase-Aktivität durch Licht beschrieben. Dieses Verfahren basiert auf Rapamycin-FKBP-induzierte FRB Heterodimerisierung und Foto einschließende Systeme. Optimierung der Licht-Bestrahlung ermöglicht die raum-zeitlich kontrollierte Aktivierung der kleinen GTPasen auf subzellulärer Ebene.

Serie: Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Fluorescence Lifetime Imaging für molekulare Rotoren in lebenden Zellen

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Forschung

Biotechnik

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Messung der mechanischen Eigenschaften von lebenden Zellen mit Atomic Force Microscopy

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Quantitative und zeitliche Steuerung der Sauerstoff-Mikromilieu in der Einzel-Islet Niveau

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Ratiometric Biosensors, dass die mitochondriale Redox Staats-und ATP in lebenden Hefezellen Messen

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolierung von primären humanen Colon Tumorzellen von Surgical Gewebe und Kultivierung sie direkt auf weiche elastische Substrate für Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Ein Markierungsfreie Technik für die Raum-zeitliche Imaging von Single Cell Sekrete

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Optische Kontrolle von lebenden Zellen elektrische Aktivität von konjugierten Polymeren

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-Kultur des Lebens Microbiome mit mikro humanen intestinalen Villi in ein Gut-on-a-Chip-Mikrofluidik-Vorrichtung

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolierung von mesenchymalen Stammzellen aus menschlichen alveoläre Periost und Effekte von Vitamin D auf knochenbildenden Aktivität Periost-abgeleitete Zellen

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Messung von Kraft-und Kleinschreibung Proteindynamik in lebenden Zellen mit einer Kombination von fluoreszierenden Techniken

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...