Vinculin-GFP plasmid was a gift from Kenneth Yamada, National Institutes of Health, National Institute of Dental and Craniofacial Research, Bethesda, Maryland, and actin-mRFP plasmid was a gift from Michael Davidson, Florida State University, Tallahassee, Florida.
This work was supported by NSF Career award #0747334 and AHA-National SDG #0835205N to Andreea Trache.

1. Overview of the Integrated Microscope System
The microscope system that is used for these studies has been described in detail 1. Briefly, an inverted Olympus IX-81 microscope with TIRF attachment (Olympus, Center Valley, PA) is combined with CSU-22 Yokogawa scanning head (Yokogawa Electric Inc, Japan). A Bioscope SZ atomic force microscope (Veeco Instruments Inc, Santa Barbara, CA) is mounted on top of the optical inverted microscope, and the entire system is placed on top of a research grade optical table (Newport, irvine, CA).
The confocal assembly composed from the spinning-disk scanner, external filter wheel and its EMCCD camera (Roper Scientific-Photometrics, Tucson, AZ) are rigidly connected with an aluminum base plate placed on a silicon pad necessary for vibration damping. The confocal scanner connects with the microscope through a flange with a treaded collar that can be decoupled from the microscope during AFM measurements, such that no vibration from the spinning-disk scanner will affect the AFM measurements. An Argon-Krypton laser (Spectra Physics, Mountain View, CA) is used as excitation source for both TIRF and confocal imaging modes, and can be coupled alternatively in two optical fibers which deliver the laser to the TIRF attachment or the scanning head, respectively. Two computers control the system: Slidebook software controls the optical imaging and Nanoscope software controls the AFM.
2. Cell Culture
Live cells expressing GFP constructs targeted against specific proteins should be placed in a glass bottom cell culture dish 24 hours prior to the mechanical stimulation experiment. On the day of the experiment replace the cell culture medium with phenol red-free medium. The cell culture dish is mounted on the AFM stage with a magnetic collar.
3. AFM Tip Preparation and Microscope Set-up
The AFM tip customized with a 2 micron biotinilated glass bead will be washed five times with DPBS and then incubated 5 min with avidin (1 mg/ml), washed again with DPBS five times, and then incubated 5 min with 1 mg/mL biotinilated fibronectin. The tip will be washed five more times at the end 2, mounted on the AFM scanner, and then the protective silicon skirt will be placed around the holder. Set the microscope for video viewing to align the AFM tip in the center of the field of view. Align the optical lever by placing the laser beam at the very end of the cantilever, and then change the objective to a high magnification objective proper for single cell imaging. The spring constant of the tip has to be determined before starting the experiment. This parameter will be measured using the thermal tune method available in the Nanoscope software. Switch from video camera to EMCCD camera to allow for florescence imaging. Then, choose a cell and bring the AFM tip in contact with that specific cell. After 20 min waiting time, which is necessary for a strong focal adhesion to form at the AFM point of contact with the cell surface, the mechanical stimulation procedure will start.
4. Mechanical Stimulation of the Live Cell
TIRF imaging 3-4 will be the method of choice for visualizing and quantifying focal adhesions, and spinning-disk confocal imaging 5 will be used for visualizing the actin cytoskeleton throughout the cell body. The AFM 6 will be used in contact mode imaging under fluid.
A TIRF or confocal image of the cell will be acquired before starting the mechanical stimulation procedure. The mechanical stimulation will be induced by the functionalized AFM probe that will be moved upward in discrete steps every 3-5 min over a period of time (1-1.5 hours). The cell reactive-response will be recorded continuously in Nanoscope software as a series of height images, each line in the image representing a unit time of 1 sec. While acquiring the AFM data, the operator monitors at all times the z-position of the AFM tip to determine the proper timing for the next pull.
After each mechanical stimulation it is possible to acquire a TIRF or confocal image of the cell in Slidebook software, while continuously acquiring AFM data. The optical images can be processed off-line to create movies that show actin cytoskeleton and focal adhesions reorganization. Also, each image can be processed quantitatively to determine cell area, specific protein area, and analyze the general cell morphology.
At the end of the experiment the AFM tip is withdrawn from the cell and AFM tip sensitivity will be measured in the Nanoscope software.
The experiment is very noise sensitive! Any talking or microscope touching during the experiment should be avoided. To minimize the noise in the AFM measurements, the spinning-disk confocal should be de-coupled from the microscope. The waiting time to form a strong focal adhesion between cell surface and the AFM tip may vary depending on the cell type used and the matrix coated on the tip.
5. Representative Results
<img src="/files/ftp_upload/2072/2072fig1.jpg" alt="Figure 1" /> Figure 1. Same vascular smooth muscle cell (VSMC) transfected with actin-mRFP and vinculin-GFP was imaged by: (A) AFM at the apical cell surface; (B) TIRF at the basal cell surface; and (C) spinning-disk confocal microscopy throughout the cell body. The confocal image is shown as maximum projection of a 3-D stack. Good overlaps between the AFM and TIRF or spinning-disk confocal images are shown in (D) and (E), respectively. Image size is 64 x 50 micron.
<img src="/files/ftp_upload/2072/2072fig2.jpg" alt="Figure 2" /> Figure 2. A VSMC transfected with actin-mRFP was mechanically stimulated by the AFM and imaged by spinning-disk confocal microscopy. The arrows indicate actin fibers that: (a) polymerize/thicken, (b) depolymerize/disappear, or (c) bundle together due to actin restructuring. White dashed lines represent the AFM tip above the cell. Image size 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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy.">Atomic Force Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.17 (2008).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J Biomed Opt. 6, 6-13 (2001).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+Internal+Reflection+Fluorescence+Microscopy.">Total Internal Reflection Fluorescence Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.22 (2008).</li><li>Inoue, S., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-view+high-speed+confocal+scanner:+the+CSU-10.">Direct-view high-speed confocal scanner: the CSU-10.</a> Methods Cell Biol. 70, 87-127 (2002).</li><li>Binnig, G., Quate, C. F., Gerber, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope.">Atomic force microscope.</a> Phys Rev Lett. 56, 930-933 (1986).</li><li>Stamenovi&#263;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+mechanics+in+airway+smooth+muscle+cells.">Cytoskeletal mechanics in airway smooth muscle cells.</a> Respir Physiol Neurobiol. 163, 25-32 (2008).</li><li>Gunst, S. J., Tang, D. D., Opazo Saez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+remodeling+of+the+airway+smooth+muscle+cell:+a+mechanism+for+adaptation+to+mechanical+forces+in+the+lung.">Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung.</a> Respir Physiol Neurobiol. 137, 151-168 (2003).</li><li>Worth, N. F., Rolfe, B. E., Song, J., Campbell, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cell+phenotypic+modulation+in+culture+is+associated+with+reorganization+of+contractile+and+cytoskeletal+proteins.">Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganization of contractile and cytoskeletal proteins.</a> Cell Motility and the Cytoskeleton. 49, 130-145 (2001).</li><li>Romer, H. R., Birukov, K. G., Garcia, J. G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+paradigm+for+a+signaling+nexus.">Focal Adhesions: paradigm for a signaling nexus.</a> Circulation Research. 98, 606-616 (2006).</li><li>Opazo Saez, A., Zhang, W., Wu, Y., Turner, C. E., Tang, D. D., Gunst, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+development+during+contractile+stimulation+of+smooth+muscle+requires+recruitment+of+paxillin+and+vinculin+to+the+membrane.">Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane.</a> Am J Physiol Cell Physiol. 286, C433-C447 (2004).</li><li>Alenghat, F. J., Ingber, D. 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. K., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+a+nexus+for+intracellular+signaling+and+cytoskeletal+dynamics.">Focal Adhesions: a nexus for intracellular signaling and cytoskeletal dynamics.</a> Exp Cell Res. 261, 25-36 (2000).</li><li>Orr, A. W., Helmke, B. P., Blackman, B. R., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mechanotransduction.">Mechanisms of mechanotransduction.</a> Dev Cell. 10, 11-20 (2006).</li></ol>

No conflicts of interest declared.

Cytoskeleton 7-10 and focal adhesions 11-16 are dynamic structures that assemble, disperse and turn over as cells migrate or respond to mechanical force 17. They have significant functions in regulating cell growth, survival, and gene expression. Knowing their specific distribution and dynamics provides a better understanding of how cells communicate with the matrix and between themselves.
This new microscope system is broadly applicable across a wide range of...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
</tr>		</tbody>
		</table>
		</div>

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

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 investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

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

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

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

Research

JoVE Journal

Biology

11.2K vistas.  Texas A&M Health Science Center. Este documento tiene como objetivo instruir al lector en la operación de una fuerza integrada atómica microscopio óptico de imagen para la estimulación mecánica de las células vivas en la cultura. Un protocolo de paso a paso se presenta. Un conjunto representativo de datos que muestra la respuesta de células vivas a la estimulación mecánica se presenta.

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

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

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

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.

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.

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.

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.

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.

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.

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.

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.

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