Cette étude a été financée par le NIH financement de la recherche (DK090868 et GM092930 à TI). Il ya une attente de brevet sur un analogue de la rapamycine en cage.

1. La transfection de l&#39;ADN plasmidique <ol><li> Ajouter ADN plasmidiques, 0,5 ug membrane captif FRB (ci-après dénommé LDR) et 0,5 ug FKBP-Tiam1 (T-cell invasion lymphome et les métastases induisant la protéine 1: Rac activateur) marqué avec la protéine fluorescente à 37,5 pi dH 2 O. Ajouter 1 ul FuGENE HD. </li><li> Vortex et incuber à température ambiante pendant 20 minutes. Pendant ce temps, procéder aux étapes 1,3-1,8. </li><li> Laver chaque puits d&#39;une chambre de 8 puits avec 50 pl de poly-D-Lysine. </li><li> Laver le centre de chaque puits avec dH 2 O. </li><li> Trypsiniser 80-85% confluentes cellules NIH-3T3 et diluer à 10 ml du milieu de culture (DMEM avec 10% de FBS). </li><li> Retirer 375 ul de la suspension et centrifuger à 1750 rpm pendant 3 minutes. </li><li> Aspirer les médias, en faisant attention de ne pas perturber le culot. </li><li> Ajouter 750 ul de FBS DMEM w / 10% et remettre en suspension. </li><li> Ajouter suspension cellulaire à dH 2 O / ADN / FuGENE HD solution et mélanger doucement. </li><li> Ajouter 250ul de cellules / ADN de suspension dans chaque puits (remplira jusqu&#39;à 3 puits). </li><li> Incuber les cellules transfectées à 37 ° C et 5% de CO 2 pendant 15-18 h. </li></ol> 2. Préparation de la CRB-Une Solution <ol><li> Synthétiser en cage rapamycine-biotine adduit (CRB). Voir «schéma de synthèse du CRB&quot; pour plus de détails. Brièvement, des fragments de cages et de la biotine sont préparés séparément. Le fragment cage est ensuite conjugué à la rapamycine (CL laboratoire), le produit ensuite couplé à la fraction biotine par un moyen de réaction 7 clic pour donner CRB. </li><li> Préparer une solution à 1 uM de stock du CRB dans le DMSO. </li><li> Dissoudre 1 mg d&#39;avidine dans 250 ul de PBS dans un tube de 0,5 ml Eppendorf. </li><li> Ajouter 1 ml de solution stock de CEM pour l&#39;avidine dissous. Mélanger en retournant plusieurs fois. </li><li> Incuber à température ambiante pendant 15 min pour obtenir CRB-avidine conjugué (CRB-A) Solution. </li><li> Exclure les petites molécules non liées et purifier CRB-A. En utilisant une colonne d&#39;exclusion de taille </li></ol> 3. Microscopie et un éclairage en lumière au niveau subcellulaire <ol><li> Sérum affamer les cellules transfectées dans du DMEM sans SVF pendant 12 heures. </li><li> Aspirer doucement les médias hors des cellules et ajouter 250 microlitres de la préparation 400 nM CRB-Une solution (de l&#39;étape 2.6). </li><li> Pour visualiser la dynamique des protéines dans les cellules vivantes à la résolution subcellulaire avant et après traitement par la lumière, la microscopie disque de rotation a été utilisé (Figure 2a). Illumination UV a été réalisée par couplage optique pour la lumière LED UV (365 nm) avec l&#39;épi-fluorescence en lumière (Figure 2b). </li><li> MetaMorph logiciel a été utilisé pour capturer des images de fluorescence des cellules transfectées toutes les 15 secondes. </li><li> Définir les coordonnées et la taille d&#39;un point d&#39;illumination en utilisant une plaque de verre revêtue avec des colorants fluorescents excités par la lumière UV. La taille peutfaire varier en appliquant lentille d&#39;objectif avec des grossissements différents et en sélectionnant des fibres optiques avec la taille de base différente. </li><li> Après la détermination des niveaux de fond de fronçage membrane, la lumière UV irradient à la périphérie de cellules. Les deux étapes suivantes sont à contraster précédant effets localisés. </li><li> Globalement irradier des cellules avec de la lumière à 365 nm fournie par une lampe à mercure. </li><li> Dissoudre Uncaged, ainsi rapamycine origine dans le DMSO, puis dans du PBS (400 nM final), et ajouter la solution à la chambre de l&#39;imagerie d&#39;induire des effets globaux. </li></ol> 4. Les résultats représentatifs Un exemple de résultats représentatifs est illustré à la figure 3. Irradiation de lumière UV à une petite région près d&#39;un bord cellulaire induit rapidement la formation de volants localisée que dans et à proximité de la zone irradiée. Pour confirmer qu&#39;il s&#39;agissait effectivement d&#39;une activité locale, nous avons alors le monde irradié des cellules avec des UV, ou ajouté rapamycine aux médias en vrac, à la fois de la WHIch induit la formation tout au long de volant mondiale de la membrane. Pour une analyse quantitative, nous avons divisé les cellules cibles en quatre quadrants, et a compté la fréquence de formation volants dans chaque quadrant sur locale ainsi que suite à une stimulation globale dans diverses conditions, y compris les contrôles (Figure 4). Ce n&#39;est que cette combinaison de l&#39;irradiation UV, CRB-A, et appropriées FKBP-FRB constructions qui induit fronçage locale. <img alt="Figure 1" src="/files/ftp_upload/3794/3794fig1.jpg" /> Figure 1. Schéma de la dimérisation des protéines dans l&#39;espace confiné à l&#39;aide en cage rapamycine-avidine conjugué (CRB-A) et de la lumière UV. Irradiation UV clive la liaison entre la rapamycine et de la biotine, entraînant la libération de produits chimiques de dimérisation (HE-Rapa, C40-O-(2-hydroxyethyl) rapamycine) et son sous-produit. La dimérisation publié diffuse ensuite dans les cellules et induit la dimérisation entre FKBP-POI (protéine d&#39;intérêt) et le plasma membrane ancré FRB que dans la proximité de la région irradiée (cercle bleu). Sans l&#39;irradiation UV, FKBP et FRB n&#39;associent pas (croix rouge), donc ne produisant aucun effet. (Copyright @ ACS2011) 6 <img alt="Figure 2" src="/files/ftp_upload/3794/3794fig2.jpg" /> Figure 2. Image du microscope personnalisé confocale de fluorescence. (A) Vue de face du microscope confocal (inversé Axiovert 200, Zeiss). (B) Close-up, vue de dessus d&#39;une unité de couplage pour le PEV de fluorescence et d&#39;illumination UV. <img alt="Figure 3" src="/files/ftp_upload/3794/3794fig3.jpg" /> Figure 3. Formation volants spatialement confinée dans un transfectée NIH3T3 cellulaire induite par une irradiation UV localisée. Confocale de fluorescence images d&#39;un cellulaire de fibroblastes NIH3T3 qui est transfectée avec YFP-étiqueté FKBP-Tiam1 (Rac activateur) et LDR (membrane ancrée FRB) a subi une irradiation dans un espace confiné près de son bord avec LED lumière à 365 nm pour 15 secondes à partir de 210 sec en présence d&#39;CRb-Conjugué dans les supports en vrac. Ce traitement a été suivi par l&#39;illumination globale avec lampe au mercure pour 1 sec à 1063 sec plus et globale de la rapamycine finales 400 nM à 1560 sec. La cellule a montré presque aucune activité ébouriffant au début (a), mais a montré dans l&#39;espace confiné ébouriffant après l&#39;illumination localisée (cercle jaune) (b). La cellule a montré la formation de volant mondiale après l&#39;illumination globale et l&#39;ajout de la rapamycine (cd), indiquant que la formation locale a été volants provoquée par l&#39;activation dans l&#39;espace confiné de Rac. La barre d&#39;échelle: 10 um. (Copyright @ ACS2011) 6 <img alt="Figure 4" src="/files/ftp_upload/3794/3794fig4.jpg" /> Figure 4. Fréquence de la formation volants dans les quadrants de cellules NIH3T3 transfectées. Irradiation UV locale CRB-A et après irradiation UV mondiale ont été réalisées sur 13 cellules. CRB-A </sTrong outre&gt; (pas d&#39;irradiation UV), une irradiation UV locale avec ou sans l&#39;avidine ont été mis en oeuvre sur des cellules 13, 5 et 6, respectivement. Aucune des cellules observées ont montré la formation de volant CRB-A sans irradiation UV, ou l&#39;irradiation UV locale avec ou sans l&#39;avidine dans les médias en vrac. Nous avons recueilli des données à partir de cellules avec la formation de fond volant bas, mais qui a la capacité de la rapamycine induit la formation de volant, ce qui nous avons vérifié après chaque expérience en confirmant la formation volants mondiale en tant que résultat de l&#39;addition de la rapamycine (finales 400 nM). Ces résultats ont montré que CRB-A et l&#39;irradiation UV locale sont nécessaires pour la formation locale volant dans les cellules. (Copyright @ ACS2011) 6 Abréviations: FKBP: FK506-binding protein FRB: FKBP-rapamycine-binding protein Tiam1: T-cell invasion lymphome et les métastases induisant protein 1 (Rac FEM) LDR: Lyn-DSAG linker-FRB (Lyn: N-terminales 11 acides aminés de Lyn kinase) HE-Rapa: C40-O - (2-hydroxyéthyl) rapamycine CRB: la rapamycine en cage avec fragment de biotine CRB-A: CRB conjugué à l&#39;avidine

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Pas de conflits d&#39;intérêt déclarés.

Nous avons décrit une technique qui emploie un roman composé en cage avec le système hétérodimérisation FKBP-FRB afin de manipuler Rho GTPase activité à un endroit précis sur une échelle subcellulaire de temps en secondes. Il ya trois limites de l&#39;approche actuelle. Tout d&#39;abord, parce que la méthode est basée sur la prévention ou permettant la diffusion des dimérisation travers la membrane plasmique, l&#39;emplacement cible cellulaire doit être la membrane plasmique o...

<table border="1"><tbody><tr><td> Nom du réactif / équipements </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> La rapamycine </td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE HD </td><td> Roche </td><td> 04709691001 </td></tr><tr><td> Avidine </td><td> Sigma </td><td> A9275-10MG </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650-5X5ML </td></tr><tr><td> Taille-exclusion colonne </td><td> GE Healthcare </td><td> Piège Spin G-25 </td></tr><tr><td> À fond de verre de 8 puits de chambre </td><td> Thermo Scientific </td><td> 12-565-47 </td></tr><tr><td> Microscope inversé à fluorescence </td><td> Zeiss </td><td> Axiovert200M </td></tr><tr><td> LED UV source de lumière </td><td> Rapp Opto Electronics </td><td> UVILED </td></tr><tr><td> Confocale disque en rotation </td><td> Yokogawa Electric Corp </td><td> CSU10 </td></tr><tr><td> La caméra CCD </td><td> Hamamatsu Photonics </td><td> ORCA-ER </td></tr><tr><td> 100x l `objectif </td><td> Zeiss </td><td> Plan Apochromat </td></tr></tbody></table> Schéma de synthèse du CRB (droit d&#39;auteur ACS2011) 6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="Schéma 1" /></a> Les conditions de synthèse: (a) BnBr, K 2 CO 3, du DMF (b) f.HNO 3, AcOH (c) TFA (d) propargyl-Br, K 2 CO 3, du DMF, (e) glycérol, le chat. pTSA, toluène (f) NaBH 3 CN, TiCl 4, MeCN (g) Tf 2 O, pyridin méthanol (h) HCl, (i) LiAlH 4, THF (j) TsCl, pyridine, CH 2 Cl 2 (k) NaN 3, DMF (l) 8, 2,6-di-t-butylpyridine, CH 2 Cl 2 (m) 13, CuSO 4, ascorbate, le 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

Régulation dynamique de la famille Rho guanosine triphosphatases petites GTPases) avec une précision spatio-temporelle est grande essentiels pour diverses fonctions cellulaires et des événements 1, 2. Leur nature spatio-temporelle dynamique a été révélé par la visualisation de leur activité et la localisation en 3 temps réel. Afin d&#39;obtenir une meilleure compréhension de leurs rôles dans diverses fonctions cellulaires au niveau moléculaire, la prochaine étape devrait être la perturbation des activités de protéines à un endroit précis subcellulaire et le calendrier. Pour atteindre cet objectif, nous avons développé une méthode pour induite par la lumière, spatio-temporellement activation contrôlée de petites GTPases en combinant deux techniques: (1) rapamycine-FKBP-FRB induite par hétérodimérisation et (2) une méthode de photo-mise en cage de la rapamycine. Avec l&#39;utilisation de la rapamycine-FKBP-FRB médiation hétérodimérisation, nous avons développé une méthode pour rapidement inductible activation ou l&#39;inactivation des petites GTPases including Rac 4, Cdc42 4, 4 RhoA et Ras 5, dans lequel la rapamycine induit la translocation de la FKBP-condensés, ou leurs GTPases activateurs, à la membrane plasmique, où FRB est ancré. Pour le couplage avec ce système hétérodimérisation, nous avons également développé un système de photo-mise en cage de la rapamycine analogues. Un composé photo-cage est une petite molécule dont l&#39;activité est supprimée avec un groupe photoclivable protéger connu sous le nom d&#39;un groupe de mise en cage. Pour supprimer l&#39;activité hétérodimérisation complètement, nous avons conçu une rapamycine en cage qui est attaché à une macromolécule de telle sorte que le complexe résultant grande ne peut pas traverser la membrane plasmique, ce qui conduit à pratiquement aucune activité de fond comme une dimérisation chimique à l&#39;intérieur des cellules 6. Figure 1 illustre un schéma de notre système. Avec la combinaison de ces deux systèmes, nous recrutés localement un activateur de Rac à la membrane plasmique sur une échelle de temps de quelques secondes et atteint induite par la lumière d&#39;activation Rac à la subcellulaire 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

Procédé pour spatio-temporel de commande de l&#39;activité GTPase de petite par la lumière est décrite. Cette méthode est basée sur la rapamycine-FKBP-FRB induite par hétérodimérisation et photo-cages systèmes. Optimisation de la lumière-l&#39;irradiation permet l&#39;activation spatio-temporellement contrôlée de petites GTPases au niveau subcellulaire.

Spatio-temporelle de manipulation de l&#39;activité GTPase Petit au niveau subcellulaire et sur des échelles de secondes dans les cellules vivantes

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

10.8K vues.  Johns Hopkins University. Procédé pour spatio-temporel de commande de l'activité GTPase de petite par la lumière est décrite. Cette méthode est basée sur la rapamycine-FKBP-FRB induite par hétérodimérisation et photo-cages systèmes. Optimisation de la lumière-l'irradiation permet l'activation spatio-temporellement contrôlée de petites GTPases au niveau subcellulaire.

Vidéo: 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.

Imagerie de vie de fluorescence des rotors moléculaires dans les cellules vivantes

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

Recherche

Bio-ingénierie

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.

Contrôle quantitatif et temporelle des micro-environnement de l&#39;oxygène à l&#39;Islet Plain

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

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.

Isolation des droits de cellules primaires de tumeurs du côlon à partir de tissus chirurgicaux et les cultivant directement sur élastique souple substrats pour Traction cytométrie

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.

Une technique Label-free pour l&#39;imagerie spatio-temporelle de cellule unique sécrétions

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.

Contrôle optique des cellules vivantes activité électrique par conjugués polymères

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-culture de la vie microbiome avec Microengineered Human Intestinal Villi dans un Gut-on-a-Chip microfluidique périphérique

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.

Isolement des cellules souches mésenchymateuses du périoste alvéolaire humaine et les effets de la vitamine D sur activité ostéogène du périoste dérivées des cellules

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

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Microscopie de Fluorescence lumière-feuille pour capturer des Images 4-dimensionnelle des effets de modulation de la contrainte de cisaillement sur le coeur de poisson-zèbre en développement

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

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.

Mesure de la dynamique des protéines sensibles à la Force dans les cellules vivantes à l’aide d’une combinaison de Techniques Fluorescent

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

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Modelage de la géométrie des colonies de cellules souches embryonnaires humaines sur des substrats conformes pour contrôler la mécanique de niveau tissulaire

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...