Questo studio è stato sostenuto da NIH finanziamento della ricerca (DK090868 e GM092930 a TI). C&#39;è un brevetto per un analogo rapamicina in gabbia.

1. La trasfezione di DNA plasmidico <ol><li> Aggiungi DNA plasmide, 0,5 mg di membrana-tethered FRB (di seguito denominato LDR) e 0,5 mcg FKBP-Tiam1 (T-cell linfoma invasione e metastasi che inducono proteina 1: Rac attivatore) codificata con una proteina fluorescente a 37,5 ul dH 2 O. Aggiungere 1 ml Fugene HD. </li><li> Vortex e incubare a temperatura ambiente per 20 minuti. Nel frattempo, procedere ai passaggi 1,3-1,8. </li><li> Lavare ciascun pozzetto di una camera 8-bene con 50 microlitri poli-D-Lisina. </li><li> Lavare il centro di ciascun pozzetto con dH 2 O. </li><li> Tripsinizzare 80-85% confluenti NIH-3T3 e diluire a 10 ml di terreno di coltura (DMEM con 10% FBS). </li><li> Rimuovere 375 microlitri di sospensione e centrifugare a 1750 rpm per 3 minuti. </li><li> Aspirare i media, facendo attenzione a non disturbare il pellet. </li><li> Aggiungere 750 microlitri di FBS DMEM w / 10% e risospendere. </li><li> Aggiungi sospensione cellulare al dH 2 O / DNA / Fugene soluzione HD e mescolare delicatamente. </li><li> Aggiungere 250microlitri di cellule / DNA sospensione a ciascun pozzetto (si riempirà fino a 3 pozzetti). </li><li> Incubare le cellule transfettate a 37 ° C e 5% CO 2 per 15-18 ore. </li></ol> 2. Preparazione del CRB-A Solution <ol><li> Sintetizzare in gabbia rapamicina-biotina addotto (CRB). Vedi &quot;Schema sintetico del CRB&quot; per i dettagli. In breve, frazioni di ingabbiamento e biotina vengono elaborati separatamente. La porzione di gabbia è poi coniugata rapamicina (LC lab), successivamente il prodotto accoppiato alla porzione biotina con un mezzo di reazione click 7 ottenendo CRb. </li><li> Preparare una soluzione stock 1 uM del CRB in DMSO. </li><li> Sciogliere 1mg avidina in 250 microlitri di PBS in una provetta Eppendorf da 0,5 ml. </li><li> Aggiungere 1 ml di soluzione madre crb di avidina dissolto. Mescolare agitando parecchie volte. </li><li> Incubare a temperatura ambiente per 15 minuti per ottenere CRB-coniugato avidina (CRB-A) Soluzione. </li><li> Escludi molecole non legate piccole e purificare crb-A utilizzando una dimensione di esclusione della colonna. </li></ol> 3. Microscopia e illuminazione luce a livello subcellulare <ol><li> Siero fame cellule trasfettate in DMEM senza FBS per 12 ore. </li><li> Aspirare delicatamente i media al largo delle celle e aggiungere 250 microlitri della preparato 400 nM crb-A soluzione (dal punto 2.6). </li><li> Per visualizzare dinamica della proteina in cellule viventi a risoluzione subcellulare prima e dopo il trattamento con luce, microscopia disco rotante è stata utilizzata (Figura 2a). Illuminazione UV è stato ottenuto per accoppiamento ottica UV LED (365 nm) con epi-fluorescenza luce (Figura 2b). </li><li> MetaMorph software è stato utilizzato per catturare le immagini di fluorescenza di cellule trasfettate ogni 15 secondi. </li><li> Definite le coordinate e la dimensione di un punto di illuminazione utilizzando una lastra di vetro rivestita con coloranti fluorescenti eccitato dalla luce UV. La dimensione puòessere variata applicando lente obiettivo con diversi ingrandimenti e scegliendo fibre ottiche con diverse dimensioni del nucleo. </li><li> Dopo la determinazione di livelli di fondo arruffarsi membrana, irradiare luce UV alla periferia delle cellule. Le seguenti due passaggi sono precedenti per contrastare gli effetti localizzati. </li><li> Globalmente irradiare le cellule con la luce a 365 nm da una lampada di mercurio. </li><li> Sciogliere Uncaged, così rapamicina originale in DMSO e poi in PBS (400 nM finale), e aggiungere la soluzione alla camera di imaging per indurre effetti globali. </li></ol> 4. Risultati rappresentativi Un esempio dei risultati rappresentativi è mostrato in Figura 3. Irradiazione di luce UV in una piccola regione in prossimità di un bordo cellulare rapidamente indotto la formazione di volant localizzata solo in e vicino alla zona irradiata. Per confermare che questo era effettivamente l&#39;attività locale, quindi a livello globale le cellule irradiate con raggi UV, o aggiunti rapamicina ai media di massa, sia di WHIch indotto formazione globale balza in tutta la membrana. Per un&#39;analisi quantitativa, abbiamo diviso le cellule bersaglio in quattro quadranti, e contava la frequenza della formazione volant in ogni quadrante sul locale così come in seguito a stimolazione globale in condizioni diverse, inclusi i controlli (Figura 4). E &#39;solo questa combinazione di irradiazione UV, la CRB-A, e adeguate FKBP-FRB costrutti che induce arruffarsi locale. <img alt="Figura 1" src="/files/ftp_upload/3794/3794fig1.jpg" /> Figura 1. Schematica di dimerizzazione proteina spazialmente confinata utilizzando gabbia rapamicina-coniugato avidina (CRB-A) e alla luce UV. Irradiazione UV scinde il linker tra rapamicina e biotina, con conseguente rilascio di chimica dimerizer (HE-Rapa, C40-O-(2-idrossietil) rapamicina) e il suo sottoprodotto. Il dimerizer rilasciato diffonde nelle cellule e induce dimerizzazione tra FKBP-POI (proteina di interesse) e plasma membRane ancorata FRB solo in prossimità della regione irradiata (cerchio blu). Senza l&#39;irradiazione UV, FKBP e FRB non associato (croce rossa), producendo quindi alcun effetto. (Copyright @ ACS2011) 6 <img alt="Figura 2" src="/files/ftp_upload/3794/3794fig2.jpg" /> Figura 2. Immagine della misura microscopio confocale a fluorescenza. (A) vista frontale del microscopio confocale (invertito Axiovert 200, Zeiss). (B) primo piano, vista dall&#39;alto di un dispositivo di accoppiamento per EPI fluorescenza e illuminazione UV. <img alt="Figura 3" src="/files/ftp_upload/3794/3794fig3.jpg" /> Figura 3. Formazione ruffle spazialmente confinati in una trasfettato NIH3T3 cellulare indotta da radiazioni UV localizzato. Confocale immagini di fluorescenza di un cellulare di fibroblasti NIH3T3 che è trasfettata con YFP-tagged FKBP-Tiam1 (Rac attivatore) e LDR (membrana ancorata FRB) ha subito l&#39;irradiazione in una zona limitata in prossimità del suo bordo con LED luce a 365 nm per 15 secondi a partire da 210 sec in presenza di CRB-A coniugato nei mezzi di bulk. Questo trattamento è stato seguito da illuminazione globale con lampada al mercurio per 1 sec a oltre 1.063 sec e globale della rapamicina finali 400 Nm a 1.560 sec. La cella ha mostrato quasi alcuna attività scompigliava all&#39;inizio (a), ma ha mostrato spazialmente limitata scompigliava dopo l&#39;illuminazione localizzata (cerchio giallo) (b). La cella ha mostrato formazione globale balza dopo l&#39;illuminazione globale e l&#39;aggiunta della rapamicina (cd), indicando che la formazione balza locale è stata causata dall&#39;attivazione di Rac spazialmente limitata. Scale bar: 10 pm. (Copyright @ ACS2011) 6 <img alt="Figura 4" src="/files/ftp_upload/3794/3794fig4.jpg" /> Figura 4. Frequenza di formazione balza in quadranti di trasfettate cellule NIH3T3. Irradiazione UV locale con CRB-A e dopo irradiazione UV globale sono stati eseguiti su cellule 13. CRB-A </sInoltre Trong&gt; (no irradiazione UV), irradiazione UV locale con o senza avidina stati attuati su cellule 13, 5 e 6, rispettivamente. Nessuna delle cellule osservate mostrato formazione crespo con CRb-A senza irradiazione UV, o irradiazione UV locale con o senza mezzi di avidina in bulk. Abbiamo raccolto i dati da cellule con la formazione di bassa ruffle sfondo, ma che ha avuto la capacità di rapamicina indotta la formazione di volant, che abbiamo controllato dopo ogni esperimento confermando la formazione globale balza a seguito di aggiunta di rapamicina (finale 400 nm). Questi risultati hanno dimostrato che CRb-A e UV irradiazione locale sono necessari per la formazione ruffle locale nelle cellule. (Copyright @ ACS2011) 6 Abbreviazioni: FKBP: FK506-binding protein FRB: FKBP-rapamicina-binding protein Tiam1: T-cell linfoma invasione e metastasi-proteina che induce 1 (Rac GEF) LDR: Lyn DSAG-linker-FRB (Lyn: N-terminale di 11 aminoacidi chinasi Lyn) HE-Rapa: C40-O - (2-idrossietil) rapamicina CRB: rapamicina in gabbia con la frazione biotina CRb-A: CRb coniugato avidina

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Non ci sono conflitti di interesse dichiarati.

Abbiamo descritta una tecnica che utilizza un nuovo composto gabbia insieme FKBP-FRB eterodimerizzazione sistema per manipolare Rho GTPasi attività in una posizione precisa subcellulare su una scala di tempo in secondi. Ci sono tre limiti dell&#39;approccio attuale. Innanzitutto, poiché il metodo si basa sulla prevenzione o consentire la diffusione del dimerizer attraverso la membrana plasmatica, la posizione bersaglio cellulare deve essere membrana plasmatica o nelle sue vicinanze. Ulteri...

<table border="1"><tbody><tr><td> Nome del reagente / apparecchiatura </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Rapamicina </td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td> Fugene HD </td><td> Roche </td><td> 04709691001 </td></tr><tr><td> Avidina </td><td> Sigma </td><td> A9275-10MG </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650-5X5ML </td></tr><tr><td> Size-esclusione colonna </td><td> GE Healthcare </td><td> Spin Trappola G-25 </td></tr><tr><td> Con fondo di vetro da 8 pozzetti camera </td><td> Thermo Scientific </td><td> 12-565-47 </td></tr><tr><td> Inverted Microscopio a fluorescenza </td><td> Zeiss </td><td> Axiovert200M </td></tr><tr><td> UV sorgente luminosa a LED </td><td> Rapp Opto Elettronica </td><td> UVILED </td></tr><tr><td> Confocale disco rotante </td><td> Yokogawa Electric Corp. </td><td> CSU10 </td></tr><tr><td> CCD della fotocamera </td><td> Hamamatsu Photonics </td><td> ORCA-ER </td></tr><tr><td> 100x lente dell&#39;obiettivo </td><td> Zeiss </td><td> Piano Apochromat </td></tr></tbody></table> Schema sintetico del 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> Condizioni sintetici: (a) BnBr, K 2 CO 3, DMF (b) f.HNO 3, AcOH (c) TFA (d) Propargyl-Br, K 2 CO 3, DMF, (e) glicerolo, cat. PTSA, toluene (f) NaBH 3 CN, TiCl 4, MeCN (g) Tf 2 O, piridin (h) metanolo HCl, (i) LiAlH 4, THF (j) TsCl, piridina, CH 2 Cl 2 (k) NaN 3, DMF (L) 8, 2,6-di-t-butilpiridina, CH 2 Cl 2 (m) 13, CuSO 4, ascorbato, 2-propanolo, 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

Regolazione dinamica della famiglia di Rho triphosphatases guanosina piccole (GTPasi) con grande precisione spazio-temporale è essenziale per varie funzioni cellulari ed eventi 1, 2. La loro natura dinamica spatiotemporally è stato rivelato attraverso la visualizzazione della loro attività e localizzazione in 3 tempo reale. Al fine di ottenere una più profonda comprensione del loro ruolo in diverse funzioni cellulari a livello molecolare, il passo successivo dovrebbe essere perturbazione delle attività della proteina in una posizione precisa subcellulare e la tempistica. Per raggiungere questo obiettivo, abbiamo sviluppato un metodo per indotto dalla luce, spazio-temporalmente l&#39;attivazione controllata di piccole GTPasi combinando due tecniche: (1) rapamicina indotta FKBP-FRB eterodimerizzazione e (2) una foto-ingabbiamento metodo di rapamicina. Con l&#39;uso di rapamicina-mediata FKBP-FRB eterodimerizzazione, abbiamo sviluppato un metodo per l&#39;attivazione rapida inducibile o inattivazione di piccole GTPasi including Rac 4, Cdc42 4, RhoA 4 e Ras 5, in cui rapamicina induce traslocazione di FKBP-fusi GTPasi, o loro attivatori, alla membrana plasmatica, dove è ancorato FRB. Per l&#39;accoppiamento con questo sistema eterodimerizzazione, abbiamo anche sviluppato un sistema di foto-ingabbiamento di analoghi della rapamicina. Un composto foto-gabbia è una piccola molecola la cui attività è soppresso con un gruppo di photocleavable protezione noto come gruppo di ingabbiamento. Per sopprimere l&#39;attività eterodimerizzazione completamente, abbiamo progettato un rapamicina gabbia che è legato ad una macromolecola tale che il complesso risultante grande non può attraversare la membrana plasmatica, portando a alcuna attività di sfondo come dimerizer chimico nelle cellule 6. La figura 1 illustra uno schema di nostra sistema. Con la combinazione di questi due sistemi, a livello locale abbiamo reclutato un attivatore Rac alla membrana plasmatica su una scala temporale di secondi e ha raggiunto indotto dalla luce l&#39;attivazione Rac al subcellulare 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

Un metodo per spazio-temporale controllo delle attività di piccola GTPasi dalla luce viene descritta. Questo metodo si basa sulla rapamicina indotta FKBP-FRB eterodimerizzazione e foto-gabbie sistemi. Ottimizzazione di irradiazione di luce permette l&#39;attivazione spazio-temporalmente controllato delle piccole GTPasi a livello subcellulare.

Manipolazione spazio-temporale di attività GTPasi Piccolo a livello subcellulare e su scala temporale di secondi nelle cellule viventi

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

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JoVE Journal

Bioengineering

10.7K Views.  Johns Hopkins University. Un metodo per spazio-temporale controllo delle attività di piccola GTPasi dalla luce viene descritta. Questo metodo si basa sulla rapamicina indotta FKBP-FRB eterodimerizzazione e foto-gabbie sistemi. Ottimizzazione di irradiazione di luce permette l'attivazione spazio-temporalmente controllato delle piccole GTPasi a livello subcellulare.

Video: 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.

Lifetime Imaging di fluorescenza di rotori molecolari nelle cellule viventi

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

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Bioingegneria

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).

Misurare le proprietà meccaniche del Living cellule utilizzando microscopia a forza atomica

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.

Controllo quantitativo e temporale di ossigeno Microenvironment al Islet Single Level

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.

Biosensori MIL Tale misura mitocondriale stato redox e ATP in cellule viventi di lievito

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.

Isolamento di primarie cellule del colon tumorali umane dai tessuti chirurgici e Coltura direttamente su più elastica e flessibile substrati per trazione citometria

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.

Una tecnica senza etichetta per l&#39;Imaging spazio-temporale delle secrezioni monocellulari

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.

Controllo ottico di cellule viventi attività elettrica da coniugati Polimeri

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-cultura del vivere Microbiome con Microengineered umana intestinale Villi in un Gut-on-a-chip microfluidico dispositivo

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.

Isolamento di cellule staminali mesenchimali da periostio alveolare umano e gli effetti della vitamina D su attività osteogenica delle cellule derivate dal periostio

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.

Misurazione delle dinamiche di sensibili alla forza della proteina in cellule viventi, utilizzando una combinazione di tecniche di fluorescenza

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