Plasmide vinculina-GFP è stato un dono di Kenneth Yamada, National Institutes of Health, National Institute of Dental Research e craniofacciale, Bethesda, Maryland, e actina-MRFP plasmide era un dono di Michael Davidson, Florida State University, Tallahassee, in Florida. Questo lavoro è stato sostenuto dal premio alla carriera NSF # 0747334 e AHA-Nazionale SDG # 0835205N di Andreea Trache.

1. Panoramica del sistema integrato di microscopio Il sistema di microscopio che viene utilizzato per questi studi è stato descritto in dettaglio 1. In breve, una rovesciata Olympus IX-81 con microscopio TIRF attaccamento (Olympus, Center Valley, PA) è combinata con CSU-22 Yokogawa testa di scansione (Yokogawa Electric Inc, Giappone). Un microscopio SZ Bioscope forza atomica (Veeco Instruments Inc, Santa Barbara, CA) è montato sulla parte superiore del microscopio ottico invertito, e l&#39;intero sistema è collocato su un tavolo grado ricerca ottici (Newport, Irvine, CA). L&#39;assemblea confocale composto da spinning-disk scanner, esterno ruota portafiltri e la sua macchina fotografica EMCCD (scientifico-Roper fotometrici, Tucson, AZ) sono rigidamente collegati con una piastra base in alluminio posta su un blocco di silicio necessario per lo smorzamento delle vibrazioni. Lo scanner confocale si collega con il microscopio attraverso una flangia con un collare ricostruiti che può essere disaccoppiato dal microscopio AFM durante le misurazioni, in modo che nessuna vibrazione dallo scanner spinning-disk influenzerà le misure AFM. Un Argon-laser Krypton (Spectra Physics, Mountain View, CA) è utilizzato come fonte di eccitazione per entrambi TIRF e modalità di imaging confocale, e possono essere accoppiati in alternativa in due fibre ottiche che offrono il laser per l&#39;attaccamento TIRF o la testa di scansione, rispettivamente . Due computer di controllo del sistema: Slidebook software controlla l&#39;imaging ottico e software Nanoscope controlla l&#39;AFM. 2. Colture Cellulari Cellule viventi che esprimono GFP costruisce mirati contro specifiche proteine ​​deve essere posto in un piatto di vetro cultura fondo cellulare 24 ore prima l&#39;esperimento stimolazione meccanica. Il giorno dell&#39;esperimento sostituire il terreno di coltura cellulare con rosso fenolo-free medio. Il piatto di coltura cellulare è montato sul palco AFM con un collare magnetico. 3. AFM Suggerimento Preparazione e Microscopio Set-up La punta AFM personalizzato con un micron 2 biotinilated perle di vetro viene lavato per cinque volte con DPBS e poi incubate 5 min con avidina (1 mg / ml), lavate di nuovo con DPBS cinque volte, e poi incubati 5 minuti con 1 mg / mL biotinilated fibronectina. La punta sarà lavato cinque volte di più alla fine 2, montata sullo scanner AFM, e poi il facciale in silicone di protezione saranno disposte intorno al titolare. Impostare il microscopio per la visualizzazione di video per allineare la punta AFM al centro del campo visivo. Allineare la leva ottica mettendo il raggio laser alla fine del cantilever, e quindi modificare l&#39;obiettivo di un obiettivo ad alto ingrandimento adeguata per l&#39;imaging di singola cellula. La costante elastica della punta deve essere determinato prima di iniziare l&#39;esperimento. Questo parametro verrà misurato con il metodo termico sintonia disponibili nel software Nanoscope. Passare dalla videocamera a fotocamera EMCCD per consentire l&#39;imaging fioritura. Poi, scegliere una cella e portare la punta AFM in contatto con quella cella specifica. Dopo 20 minuti il ​​tempo di attesa, che è necessario per una forte adesione focale per formare nel punto di AFM di contatto con la superficie cellulare, la procedura di stimolazione meccanica si avvia. 4. La stimolazione meccanica del Live Cell TIRF 3-4 di imaging sarà il metodo di scelta per la visualizzazione e la quantificazione adesioni focali, e la filatura del disco di imaging confocale 5 sarà utilizzato per la visualizzazione del citoscheletro di actina in tutto il corpo cellulare. L&#39;AFM 6 sarà utilizzato nella diagnostica per immagini in modalità di contatto del liquido. Un&#39;immagine TIRF o confocale della cellula sarà acquisito prima di iniziare la procedura di stimolazione meccanica. La stimolazione meccanica saranno indotti dalla sonda funzionalizzata AFM che verrà spostato verso l&#39;alto in passi discreti ogni 3-5 minuti per un periodo di tempo (1-1.5 ore). La cellula-reattiva risposta sarà registrata in modo continuo nel software Nanoscope come una serie di immagini di altezza, ogni riga dell&#39;immagine che rappresenta una unità di tempo di 1 sec. Durante l&#39;acquisizione dei dati AFM, l&#39;operatore controlla in ogni momento la posizione z della punta AFM per determinare la tempistica adeguata per la spinta successiva. Dopo ogni stimolazione meccanica è possibile acquisire un&#39;immagine TIRF o confocale della cellula in Slidebook software, mentre continua l&#39;acquisizione di dati AFM. Le immagini ottiche possono essere elaborati off-line per creare filmati che mostrano citoscheletro di actina e focale riorganizzazione aderenze. Inoltre, ogni immagine può essere elaborata quantitativamente per determinare l&#39;area delle cellule, l&#39;area specifica proteina, e analizzare la morfologia generale delle cellule. Alla fine dell&#39;esperimento viene ritirata la punta AFM dalla cella e la sensibilità punta AFM sarà misurata nel software Nanoscope. L&#39;esperimento è molto sensibili al rumore! Qualsiasi parlare o microscopio toccando durante la experiment dovrebbe essere evitato. Per ridurre al minimo il rumore nelle misure AFM, la filatura-disk confocale dovrebbe essere disaccoppiato dal microscopio. Il tempo di attesa per formare una forte adesione focale tra la superficie delle cellule e la punta AFM può variare a seconda del tipo di cellule e la matrice rivestito sulla punta. 5. Rappresentante Risultati <img src="/files/ftp_upload/2072/2072fig1.jpg" alt="Figura 1" /> Figura 1. Stesso delle cellule muscolari lisce vascolari (VSMC) transfettate con l&#39;actina-MRFP e vinculina-GFP è stato ripreso da: (A) AFM sulla superficie cellulare apicale; (B) TIRF sulla superficie delle cellule basali, e (C) spinning-disk microscopia confocale in tutto il corpo cellulare. L&#39;immagine confocale è indicato come proiezione massima di un 3-D stack. Buone le sovrapposizioni tra AFM e TIRF o immagini confocale spinning-disk sono mostrati in (D) e (E), rispettivamente. Dimensione dell&#39;immagine è 64 x 50 micron. <img src="/files/ftp_upload/2072/2072fig2.jpg" alt="Figura 2" /> Figura 2. Un VSMC transfettate con l&#39;actina-MRFP era meccanicamente stimolato dal AFM e ripreso da spinning-disk microscopia confocale Le frecce indicano le fibre di actina che:. (A) polimerizzare / addensare, (b) depolymerize / sparire, o (c) Bundle insieme a causa alla ristrutturazione di actina. Linee bianche tratteggiate rappresentano la punta AFM sopra la cella. Dimensione dell&#39;immagine 91 x 91 micron.

<ol>
	<li>Trache, A., Lim, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microscopy+for+real-time+imaging+of+mechanotransduction+studies+in+live+cells.">Integrated microscopy for real-time imaging of mechanotransduction studies in live cells.</a> J Biomed Opt. 14, 034024-03 (2009).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction:+all+signals+point+to+cytoskeleton,+matrix+and+integrins.">Mechanotransduction: all signals point to cytoskeleton, matrix and integrins.</a> Sciences STKE. 119, pe6-pe6 (2002).</li><li>Zamir, E., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+complexity+and+dynamics+of+cell-matrix+adhesions.">Molecular complexity and dynamics of cell-matrix adhesions.</a> J Cell Sci. 114, 3583-3590 (2001).</li><li>Geiger, B., Bershdsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+mechanosensory+function+of+focal+contacts.">Assembly and mechanosensory function of focal contacts.</a> Curr Opin Cell Biol. 13, 584-592 (2001).</li><li>Sastry, S. 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Nessun conflitto di interessi dichiarati.

Aderenze citoscheletro 7-10 e 11-16 focali sono strutture dinamiche che assemblano, si disperdono e girare come le cellule migrano o rispondere a forza meccanica 17. Essi hanno funzioni importanti nella regolazione della crescita cellulare, la sopravvivenza, e l&#39;espressione genica. Conoscendo la loro distribuzione e le dinamiche specifiche fornisce una migliore comprensione di come le cellule comunicano con la matrice e tra loro. Questo sistema nuovo microscopio è ampiamente applicabile per una vasta gamma di studi di dinamica molecolare in alcun cellule aderenti vivo. Il microscopio integrato consente il monitoraggio quantitativo di rapidi cambiamenti sub-cellulare e fornisce un importante strumento innovativo per la comprensione del processo fondamentale di rimodellamento cellulare in risposta alla forza meccanica.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>AFM tip</td>
<td>&nbsp;</td>
<td>Novascan Technologies</td>
<td>&nbsp;</td>
<td>2 micron Borosilicate bead, 0.01N/m, Biotin Surface</td>
</tr>
<tr>
<td>avidin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A9275</td>
<td>1mg/mL in DPBS</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>F4759</td>
<td>1 mg/mL</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21600-051</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa transfection kit for Smooth Muscle Cells</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPI-1004</td>
<td>&nbsp;</td>
<tr>
<td>Mattek glass bottom dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P60G-1.5-30-F</td>
<td>Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5</td>
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live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy

Per capire il meccanismo con cui le cellule viventi senso forze meccaniche, e come rispondono e si adattano al loro ambiente, una nuova tecnologia in grado di studiare il comportamento delle cellule a livello sub-cellulare di livello ad alta risoluzione spaziale e temporale è stato sviluppato. Così, un microscopio a forza atomica (AFM) è stato integrato con la riflessione interna totale fluorescenza (TIRF) microscopia e in rapida rotazione del disco (FSD) microscopia confocale. Il sistema integrato è ampiamente applicabile per una vasta gamma di studi di dinamica molecolare in alcun cellule aderenti vivere, permettendo diretta imaging ottico delle risposte delle cellule alla stimolazione meccanica in tempo reale. Riorganizzazione significativa dei filamenti di actina e adesioni focali è stato dimostrato a causa di locali stimolazione meccanica sulla superficie cellulare apicale che hanno indotto cambiamenti nella struttura cellulare in tutto il corpo cellulare. Queste tecniche innovative forniranno nuove informazioni per comprendere la ristrutturazione delle cellule vive e dinamiche in risposta alla forza meccanica. Un protocollo dettagliato ed un set di dati rappresentativi che la risposta delle cellule spettacolo dal vivo allo stimolo meccanico sono presentati.

Watch this Scientific Journal Video about Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy at JoVE.com

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Questo lavoro si propone di istruire il lettore al funzionamento di un sistema integrato di forza atomica-microscopio ottico di imaging per la stimolazione meccanica delle cellule vive nella cultura. Un passo-passo il protocollo viene presentato. Un insieme rappresentativo di dati che mostra dal vivo risposta alla stimolazione meccanica delle cellule è presentato.

Risposta cellulare allo stimolo meccanico vivere Studiato da Integrated ottica e microscopia a forza atomica

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

live-cell-response-to-mechanical-stimulation-studied-integrated

Nanyang Technological University

Research

JoVE Journal

Biology

11.2K Views.  Texas A&M Health Science Center. Questo lavoro si propone di istruire il lettore al funzionamento di un sistema integrato di forza atomica-microscopio ottico di imaging per la stimolazione meccanica delle cellule vive nella cultura. Un passo-passo il protocollo viene presentato. Un insieme rappresentativo di dati che mostra dal vivo risposta alla stimolazione meccanica delle cellule è presentato.

Video: Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contrasto di fase e interferenza differenziale contrasto (DIC) Microscopia

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Ricerca

Biologia

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Micro-Caratterizzazione meccanica del tessuto polmonare usando la microscopia a forza atomica

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

La stimolazione meccanica di condrociti-agarosio idrogel

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Preparazione del campione per microscopia a forza atomica Virion singola e imaging a fluorescenza a super risoluzione

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Misurare la rigidità<em&gt; Ex Vivo</em&gt; Mouse aorte Utilizzando microscopia a forza atomica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Analizzando cellulare superficie di adesione ritocca in risposta alla tensione meccanica utilizzando biglie magnetiche

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Misurazione e risposta alla somministrazione di glucosio alla stimolazione dell&#39;insulina in<em&gt; In vitro</em&gt; Myotubes primario umano coltivato

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Valutazione della risposta di Dictyostelium discoideum alla stimolazione meccanica acuta

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

Microscopia a forza protrusione: Un metodo per quantificare le forze sviluppate dalla cella sporgenze

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...