Plazmid Vinculin-GFP Michael Davidson, Florida Eyalet Üniversitesi, Tallahassee, Florida&#39;daki bir hediye plazmid Ulusal Sağlık Enstitüleri, Ulusal Diş ve Kraniofasial Araştırma Enstitüsü, Bethesda, Maryland ve aktin-MRFP Kenneth Yamada bir hediye oldu. Bu çalışma, NSF Kariyer Ödülü # 0747334 ve AHA-Milli SDG # 0835205N Andreea Trache tarafından desteklenen oldu.

1. Entegre Mikroskop Sistemi Genel Bakış Bu çalışmalar için kullanılan mikroskop sistemi 1 ayrıntılı tarif edilmiştir. Kısaca, TIRF eki (Olympus, Center Valley, PA) ile ters bir Olympus IX-81 mikroskop ile birlikte CSU-22 Yokogawa tarama kafası (Yokogawa Elektrik A.Ş., Japonya). Bioscope SZ atomik kuvvet mikroskobu (Veeco Instruments Inc, Santa Barbara, CA), optik ters mikroskop üstüne monte edilir ve tüm sistemi bir araştırma notu optik tablonun üst (Newport, Irvine, CA) üzerine yerleştirilir. Konfokal montaj dönen disk tarayıcı, dış filtre çarkı ve EMCCD kamera (Roper Bilimsel Photometrics, Tucson, AZ) oluşan katı titreşim sönümleme için gerekli bir silikon pad üzerine yerleştirilen alüminyum taban plakası ile bağlantılıdır. Konfokal tarayıcı oluklu bir yaka ile AFM ölçümleri sırasında mikroskop ayrılabilir bir flanş ile mikroskop ile bağlanır dönen disk tarayıcı hiçbir titreşim AFM ölçümleri etkileyecektir. Bir Argon-Kripton lazer (Spectra Fizik, Mountain View, CA), sırasıyla TIRF ve konfokal görüntüleme modları için uyarma kaynağı olarak kullanılır ve alternatif TIRF eki veya tarama kafası lazer sağlayan iki optik fiberler akuple edilebilir . İki bilgisayar sisteminin kontrolü: Slidebook yazılımı optik görüntüleme kontrolleri ve NanoScope yazılım AFM kontrol. 2. Hücre Kültürü Canlı hücreler, belirli proteinlere karşı hedeflenen GFP yapıları ifade mekanik stimülasyonu deneyi 24 saat önce bir bardak alt hücre kültürü çanak konulmalıdır. Deney günü, fenol red-free orta ile hücre kültür ortamı değiştirin. Hücre kültürü çanak manyetik bir yaka ile AFM sahne üzerine monte edilmiştir. 3. AFM İpucu hazırlanması ve Mikroskop Set-up 2 mikron ile özelleştirilmiş AFM ucu, cam boncuk DPBS beş kez yıkanmış ve daha sonra 5 dakika avidin (1 mg / ml) ile inkübe, DPBS beş kez tekrar yıkanır ve daha sonra 1 mg / ml biotinilated ile 5 dakika inkübe olacak biotinilated fibronektin. Sonunda 2 ucu, beş kez daha yıkanarak AFM tarayıcı üzerine monte edilmiş ve daha sonra koruyucu silikon etek sahibinin etrafında alınacaktır olacak. Görüş alanının merkezinde AFM ucu hizalamak için video görüntüleme için mikroskop ayarlayın. Konsolun en sonunda lazer ışını koyarak optik kolu hizalayın ve sonra tek hücre görüntüleme için uygun bir yüksek büyütme amacı hedefi değiştirmek. Ucu sürekli bahar, deney başlamadan önce belirlenmelidir. Bu parametre NanoScope yazılım mevcuttur termal ayar yöntemi kullanılarak ölçülür. Floresans görüntüleme için izin EMCCD kamera, video kamera geçin. Sonra, bir hücre seçin ve bu belirli bir hücre ile temas AFM ucu getirmek. Hücre yüzeyi ile temas AFM noktada oluşturmak için güçlü bir odak yapışma için gerekli 20 dakika bekleme süresinden sonra, mekanik stimülasyonu prosedürü başlayacak. 4. Canlı Hücre Mekanik Uyarım 3-4 TIRF görüntüleme odak yapışıklıklar görselleştirilmesi ve ölçülmesi için tercih edilen bir yöntem olacak, ve dönen disk konfokal görüntüleme 5 hücre gövdesi boyunca aktin hücre iskeletinin görselleştirmek için kullanılacaktır. AFM 6 sıvı temas modunda görüntüleme kullanılır. Hücre TIRF veya konfokal görüntü mekanik stimülasyonu işlemine başlamadan önce alınacaktır. (1-1,5 saat) bir süre içinde her 3-5 dakikada bir ayrık adımlarla yukarı doğru hareket olacaktır Fonksiyonlu AFM prob mekanik stimülasyonu ile indüklenen olacaktır. Hücre reaktif-yanıt yüksekliği görüntü, görüntü temsil eden 1 sn bir birim zamanda her satırı bir dizi olarak NanoScope yazılım sürekli olarak kaydedilecektir. AFM veri elde edilirken, operatör sonraki çekme için uygun zamanlama her zaman AFM ucu z-pozisyon belirlemek için izler. Her mekanik stimülasyonu sonra sürekli AFM veriler elde edilirken, Slidebook yazılımı hücre TIRF veya konfokal görüntü elde etmek mümkündür. Aktin hücre iskeleti ve odak yapışıklıklar yeniden yapılanma gösteren filmler optik görüntü oluşturmak için off-line işlenebilir. Ayrıca, her görüntü hücre alanı, spesifik protein alanı belirlemek ve genel hücre morfolojisi analiz etmek için kantitatif olarak işlenmiş olabilir. Deneyin sonunda AFM ucu hücre ve AFM ucunun hassasiyeti çekilen NanoScope yazılım ölçülecektir. Deney çok gürültüye duyarlı! Eski sırasında dokunmadan herhangi bir konuşmaya ya da mikroskopdeneyimlemek önemlidir kaçınılmalıdır. AFM ölçümlerde gürültü en aza indirmek için, dönen disk konfokal mikroskop de çiftli olmalıdır. Hücre yüzeyi ve AFM ucu arasında güçlü bir fokal adezyon oluşturmak için bekleme süresi, kullanılan hücre tipi ve ucunda matris kaplamalı bağlı olarak değişebilir. 5. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2072/2072fig1.jpg" alt="Şekil 1" /> Şekil 1. Aktin-MRFP ve vinculin-GFP ile transfekte aynı damar düz kas hücre (VSMC) tarafından görüntülenmiş oldu: (A) apikal hücre yüzeyinde AFM, (B) bazal hücre yüzeyinde TIRF; ve (C) iplik disk konfokal mikroskopi hücre gövdesi boyunca konfokal görüntü 3-D bir yığın maksimum projeksiyon olarak gösterilmiştir . AFM ve TIRF veya dönen disk konfokal görüntüler arasında iyi örtüşür, (D) ve (E), sırasıyla gösterilmiştir . Görüntü boyutu 64 x 50 mikron. <img src="/files/ftp_upload/2072/2072fig2.jpg" alt="Şekil 2" /> Şekil 2. Bir vasküler düz kas aktin-MRFP ile transfekte mekanik AFM tarafından uyarılan ve iplik disk konfokal mikroskopi tarafından görüntülendi oklar aktin liflerinin gösterir: (a) kalınlaşmasına / Polimerize, (b) depolymerize / kaybolurken, veya (c) nedeniyle bir araya toplamak aktin yeniden yapılanma. Beyaz kesik çizgiler hücrenin üstündeki AFM ucu temsil eder. Görüntü boyutu 91 x 91 mikron.

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Sitoiskelet 7-10 ve odak yapışıklıklar 11-16, montajı, dağıtmak ve hücreleri göç veya mekanik güç, 17 cevap olarak teslim dinamik yapılardır . Hücre büyümesi, hayatta kalma ve gen ekspresyonu düzenlenmesinde önemli işlevleri var. Kendi özel dağıtım ve dinamikleri bilmek hücreleri matrisi ve kendi aralarında nasıl iletişim daha iyi anlaşılmasını sağlar. Bu yeni mikroskop sistemi yapışık herhangi bir canlı hücreler molekül...

<table><tbody><tr><td>AFM tip</td><td>Novascan Technologies, Inc.</td><td></td><td>2 micron Borosilicate bead, 0.01N/m, Biotin Surface</td></tr><tr><td>avidin</td><td>Sigma-Aldrich</td><td>A9275</td><td>1mg/mL in DPBS</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F4759</td><td>1 mg/mL</td></tr><tr><td>DPBS</td><td>Invitrogen</td><td>21600-051</td><td></td></tr><tr><td>Amaxa transfection kit for Smooth Muscle Cells</td><td>Lonza Inc.</td><td>VPI-1004</td><td></td></tr><tr><td>Mattek glass bottom dishes</td><td>MatTek Corp.</td><td>P60G-1.5-30-F</td><td>Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5</td></tr></tbody></table>

live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy

Canlı hücreler mekanik kuvvetlerin anlamda mekanizmasını anlamak ve alt hücresel düzeyde yüksek mekansal ve zamansal çözünürlüğe sahip hücrelerin davranışını araştırmak için tepki ve çevreleriyle uyum nasıl yeni bir teknoloji geliştirildi. Böylece, bir atomik kuvvet mikroskobu (AFM), toplam iç yansıma floresan (TIRF) mikroskopi ve hızlı dönen disk (FSD) konfokal mikroskobu ile entegre edildi. Entegre bir sistem, gerçek zamanlı mekanik uyarılması hücre yanıtları doğrudan optik görüntüleme sağlayan geniş yapışık herhangi bir canlı hücreler moleküler dinamik çalışmaların geniş bir aralığında uygulanabilir. Aktin filamentler ve odak yapışıklıklar önemli düzenlenmesi nedeniyle apikal hücre yüzeyinde hücre gövdesi boyunca hücresel yapıya değişikliklerin neden olduğu yerel mekanik stimülasyonu gösterildi. Bu yenilikçi teknikler, mekanik güç, cevap canlı hücre yeniden yapılanma ve dinamikleri anlamak için yeni bilgiler sağlayacaktır. Ayrıntılı bir protokol ve temsil eden bir veri mekanik stimülasyonu göstermek için canlı hücre yanıtı verildikleri ayarlayın.

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

Bu kağıt kültürü canlı hücreleri mekanik uyarılması için entegre bir atom operasyon okuyucu kuvvet optik görüntüleme mikroskop öğretmeyi hedefliyor. Bir adım-adım protokol sunulmuştur. Mekanik stimülasyonu canlı hücre yanıtı gösteren bir temsilci veri seti sunulmaktadır.

Tümleşik Optik ve Atomik Kuvvet Mikroskobu eğitimi Mekanik Uyarım Canlı Hücre Tepki

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K Görüntüleme.  Texas A&M Health Science Center. Bu kağıt kültürü canlı hücreleri mekanik uyarılması için entegre bir atom operasyon okuyucu kuvvet optik görüntüleme mikroskop öğretmeyi hedefliyor. Bir adım-adım protokol sunulmuştur. Mekanik stimülasyonu canlı hücre yanıtı gösteren bir temsilci veri seti sunulmaktadır.

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

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).

Atomik Kuvvet Mikroskobu kullanarak Yaşam Hücrelerinin Mekanik Özellikleri Ölçme

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

Biyomühendislik

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Mekanik Stretch sırasında Canlı Hücre Görüntüleme

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

Atomik Kuvvet Mikroskobu Konsol Tabanlı Nanogirinti: Hava ve Sıvıda Nanoölçekte Mekanik Özellik Ölçümleri

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Mühendislik

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

Optik Cımbız Kullanarak Hapsetmede Hücre Altı Mekaniğinin Doğrudan Kuvvet Ölçümleri

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Gelişim Biyolojisi

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Fazlararası Hücre Çekirdek Mekanik Özellikleri Probe Biyofiziksel Tahliller: Yüzey Gerilme Uygulama ve Microneedle Manipülasyon

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Biyoloji

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

Yüzlerce C. albicans Hücresinde Biyo-Atomik Kuvvet Mikroskop Ölçümlerinin Otomasyonu

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Canlı Hücrelerde Eşzamanlı Lazer Tarama Konfokal ve Atomik Kuvvet Mikroskobu (Conpokal) Yakınında

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Tek-T hücreleri veya tek parçacık Immünolojik tek hücreli kuvvet spektroskopisi ile Atomic Force mikroskop Cantilevers functionalization

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

İmmünoloji ve Enfeksiyon

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Hücre Tek katmanlar mekanik uyarılması bağlı Kalsiyum Dalga Yayılım: Sığır Kornea Endotel Hücreleri Örneği

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

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.

Çıkıntı kuvvet mikroskobu: Hücre çıkıntılar tarafından geliştirilen kuvvetleri ölçmek için bir yöntem

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