This work was supported by National Eye Institute/NIH grant R01EY028242 (to K.G.), Research to Prevent Blindness/International Retinal Research Foundation Catalyst Award for Innovative Research Approaches for AMD (to K.G.), The Stephen Ryan Initiative for Macular Research (RIMR) Special Grant from W.M. Keck Foundation (to Doheny Eye Institute), and Ursula Mandel Fellowship and UCLA Graduate Council Diversity Fellowship (to I.S.T.). This work was also supported by an Unrestricted Grant from Research to Prevent Blindness, Inc. to the Department of Ophthalmology at UCLA.&#160;The content in this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Measurement of Stiffness in Mouse Retinal Capillaries and Subendothelial Matrix

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steps+in+Mechanotransduction+pathways+that+control+cell+morphology.">Steps in Mechanotransduction pathways that control cell morphology.</a> Annu Rev Physiol. 81, 585-605 (2019).</li><li>Pan, Z., Ghosh, K., Liu, Y., Clark, R. A., Rafailovich, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+stresses+and+translational+distortion+of+the+nucleus+during+fibroblast+migration+on+a+physiologically+relevant+ECM+mimic.">Traction stresses and translational distortion of the nucleus during fibroblast migration on a physiologically relevant ECM mimic.</a> Biophys J. 96 (10), 4286-4298 (2009).</li><li>Ghosh, K., Ingber, D. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence+increases+choroidal+endothelial+stiffness+and+susceptibility+to+complement+injury:+Implications+for+choriocapillaris+loss+in+AMD.">Senescence increases choroidal endothelial stiffness and susceptibility to complement injury: Implications for choriocapillaris loss in AMD.</a> Invest Ophthalmol Vis Sci. 57 (14), 5910-5918 (2016).</li><li>Cabrera, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell+stiffness+contributes+to+complement-mediated+injury+of+choroidal+endothelial+cells+in+a+monkey+model+of+early+age-related+macular+degeneration.">Increased cell stiffness contributes to complement-mediated injury of choroidal endothelial cells in a monkey model of early age-related macular degeneration.</a> J Pathol. 257 (3), 314-326 (2022).</li><li>Krieg, M., et al. <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-based+mechanobiology.">Atomic force microscopy-based mechanobiology.</a> Nat Rev Phys. 1, 41-57 (2019).</li><li>Chou, J. C., Rollins, S. D., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+digest+protocol+to+analyze+the+retinal+vasculature+of+a+mouse+model.">Trypsin digest protocol to analyze the retinal vasculature of a mouse model.</a> J Vis Exp. (76), e50489 (2013).</li><li>Beacham, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+extracellular+matrices+produced+by+cultured+and+primary+fibroblasts.">Preparation of extracellular matrices produced by cultured and primary fibroblasts.</a> Current protocols in cell biology. , (2007).</li><li>Bae, Y. H., Liu, S. L., Byfield, F. J., Janmey, P. A., Assoian, 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=Measuring+the+stiffness+of+ex+vivo+mouse+aortas+using+atomic+force+microscopy.">Measuring the stiffness of ex vivo mouse aortas using atomic force microscopy.</a> J Vis Exp. (116), e54630 (2016).</li><li>Huynh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+intimal+stiffening+enhances+endothelial+permeability+and+leukocyte+transmigration.">Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration.</a> Sci Transl Med. 3 (112), 112 (2011).</li><li>Sharma, A., Gupta, D. K., Bisen, S., Singh, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+trypsin+and+elastase+digestion+techniques+for+isolation+of+murine+retinal+vasculature.">Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature.</a> Microvasc Res. 154, 104682 (2024).</li><li>Chaqour, B., et al. <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-based+measurements+of+retinal+microvessel+stiffness+in+mice+with+endothelial-specific+deletion+of+CCN1.">Atomic force microscopy-based measurements of retinal microvessel stiffness in mice with endothelial-specific deletion of CCN1.</a> Methods Mol Biol. 2582, 323-334 (2023).</li><li>Ashraf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+density+of+deep,+intermediate+and+superficial+vascular+plexuses+are+differentially+affected+by+diabetic+retinopathy+severity.">Vascular density of deep, intermediate and superficial vascular plexuses are differentially affected by diabetic retinopathy severity.</a> Invest Ophthalmol Vis Sci. 61 (10), 53 (2020).</li><li>Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+D:+an+Macrophage-derived+factor+mediating+increased+endothelial+cell+permeability+with+implications+for+alteration+of+the+blood-retinal+barrier+in+diabetic+retinopathy.">Cathepsin D: an Macrophage-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.</a> FASEB J. 30 (4), 1670-1682 (2016).</li></ol>

The authors have nothing to disclose.

AFM has been widely used to measure disease-associated changes in the stiffness of larger vessels, such as the aorta and arteries16. These findings have helped establish the role of endothelial mechanobiology in cardiovascular complications such as atherosclerosis17. Based on these findings, we have begun to investigate the previously unrecognized role of endothelial mechanobiology in the development of retinal microvascular lesions in early DR. Success in this pursuit, how...

Retinal capillaries are essential for maintaining retinal homeostasis and visual function. Indeed, their degeneration in early diabetes is strongly implicated in the development of vision-threatening complications of diabetic retinopathy (DR), a microvascular condition that affects nearly 40% of all individuals with diabetes1. Vascular inflammation contributes significantly to retinal capillary degeneration in DR. Past studies have demonstrated an important role for aberrant molecular and biochemical cues in diabetes-induced retinal vascular inflammation2,3. However, recent work has introduced a new paradigm for DR pathogenesis that identifies retinal capillary stiffening as a crucial yet previously unrecognized determinant of retinal vascular inflammation and degeneration4,5,6.
Specifically, the diabetes-induced increase in retinal capillary stiffness is caused by the upregulation of collagen crosslinking enzyme lysyl oxidase (LOX) in retinal endothelial cells (ECs), which stiffens the subendothelial matrix (basement membrane)4,5,6. Matrix stiffening, in turn, stiffens the overlying retinal ECs (due to mechanical reciprocity), thus leading to the overall increase in retinal capillary stiffness4. Crucially, this diabetes-induced retinal capillary stiffening alone can promote retinal EC activation and inflammation-mediated EC death. This mechanical regulation of retinal EC defects can be attributed to altered endothelial mechanotransduction, the process by which mechanical cues are converted into biochemical signals to produce a biological response7,8,9. Importantly, altered EC&#160;mechanical cues and subendothelial matrix structure have also been implicated in choroidal vascular degeneration associated with early age-related macular degeneration (AMD)10,11,12, which attests to the broader implications of vascular mechanobiology in degenerative retinal diseases.
Notably, retinal capillary stiffening occurs early on in diabetes, which coincides with the onset of retinal inflammation. Thus, the increase in retinal capillary stiffness may serve as both a therapeutic target and an early diagnostic marker for DR. To this end, it is important to obtain reliable and direct stiffness measurements of retinal capillaries and subendothelial matrix. This can be achieved by using an atomic force microscope (AFM), which offers a unique, sensitive, accurate, and reliable technique to directly measure the stiffness of cells, extracellular matrix, and tissues13. An AFM applies minute (nanoNewton-level) indentation force on the sample whose stiffness determines the extent to which the indenting AFM cantilever bends- the stiffer the sample, the more the cantilever bends, and vice versa. We have used AFM extensively to measure the stiffness of cultured endothelial cells, subendothelial matrices, and isolated mouse retinal capillaries4,5,6,11,12. These AFM stiffness measurements have helped identify endothelial mechanobiology as a key determinant of DR and AMD pathogenesis. To help broaden the scope of mechanobiology in vision research, here we provide a step-by-step guide on the use of AFM for stiffness measurements of isolated mouse retinal capillaries and subendothelial matrix.

<table><tbody><tr><td>&lt;strong&gt;Retinal Capillary Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;micro;m PVDF syringe filter</td><td>Merck Millipore</td><td>SLGVM33RS</td><td>Low Protein Binding Durapore</td></tr><tr><td>10X Dulbecco&#039;s Phosphate Buffered Saline without calcium % magnesium</td><td>Corning</td><td>20-031-CV</td><td>Final concentration 1X, pH 7.4</td></tr><tr><td>12-well plate</td><td>Falcon Corning</td><td>353043</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Corning</td><td>430791</td><td>Rnase-Dnase-free, Nonpyrogenic</td></tr><tr><td>20 mL Luer-Lok TIP syringe</td><td>BD</td><td>302830</td><td></td></tr><tr><td>5 3/4 inch Disposable Borosilicate Glass/Non-sterile Pasteur pipette</td><td>FisherBrand</td><td>13-678-20A</td><td></td></tr><tr><td>60x15 mm Tissue Culture Dish</td><td>Falcon Corning</td><td>353002</td><td></td></tr><tr><td>6-well plate</td><td>Falcon Corning</td><td>353046</td><td></td></tr><tr><td>Aqua-Hold 2 Pap - 13 mL Pen</td><td>Scientific Device Laboratory</td><td>9804-02</td><td></td></tr><tr><td>Blade holder</td><td>X-ACTO</td><td></td><td></td></tr><tr><td>Carbon Steel Surgical Blade #10</td><td>Bard-Parker</td><td>371110</td><td></td></tr><tr><td>Dental Wax</td><td>Electron Microscopy Sciences</td><td>50-949-027</td><td></td></tr><tr><td>Dissecting microscope</td><td>Am-scope</td><td></td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Millipore Sigma</td><td>HT501128-4L</td><td>Final concentration 5% (v/v)</td></tr><tr><td>Kimwipes - wiper tissue</td><td>Kimtech Science</td><td>34133</td><td></td></tr><tr><td>Micro spatula</td><td>Fine Science Tools</td><td>10089-11</td><td></td></tr><tr><td>Orbital Shaker</td><td>Lab Genius</td><td>SK-O180</td><td></td></tr><tr><td>PELCO Economy #7 Stainless Steel 115mm&amp;nbsp; Tweezer</td><td>Ted Pella, Inc.</td><td>5667</td><td></td></tr><tr><td>Phase contrast microscope</td><td>Nikon TS2</td><td></td><td></td></tr><tr><td>Purifier Logic+ Class II, Type A2 Biosafety Cabinet</td><td>Labconco</td><td>302380001</td><td></td></tr><tr><td>Safe-Lock microcentrifuge tubes 2 mL</td><td>Eppendorf</td><td>22363352</td><td></td></tr><tr><td>Stereoscope</td><td>AmScope</td><td>SM-3 Series Zoom Trinocular Stereomicroscope 3.5X-90X</td><td></td></tr><tr><td>Superfrost Plus microscopy slide - White tab - Pre-cleaned - 25x75x1.0 mm</td><td>FisherBrand</td><td>1255015</td><td></td></tr><tr><td>Tris Buffer, 0.1M solution, pH 7.4 - Biotechnology Grade</td><td>VWR</td><td>E553-500ML</td><td>pH 8 for trypsin solution</td></tr><tr><td>Trypsin 1:250 powder Tissue Culture Grade</td><td>VWR</td><td>VWRV0458-25G</td><td>10 % (w/v) trypsin solution</td></tr><tr><td>Water Molecular Biology Grade</td><td>Corning</td><td>46-000-CM</td><td></td></tr><tr><td>&lt;strong&gt;Subendothelial Matrix&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X PBS</td><td>Corning</td><td>20-031-CV</td><td></td></tr><tr><td>1X PBS with calcium and magnesium</td><td>Thermo Fisher Scientific</td><td>14040-117</td><td>pH 7.4</td></tr><tr><td>Ammonium hydroxide</td><td>Sigma-Aldrich</td><td>338818</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A4034</td><td></td></tr><tr><td>Collagen IV antibody</td><td>Novus Biologicals</td><td>NBP1-26549</td><td></td></tr><tr><td>DNase I</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>398136</td><td></td></tr><tr><td>Fibronectin antibody</td><td>Sigma-Aldrich</td><td>F6140</td><td></td></tr><tr><td>Fluoromount</td><td>Invitrogen-Thermo Fisher Scientific</td><td>00-4958-02</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Glass coverslips (12mm)</td><td>Fisher</td><td>12-541-000</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Electron microscopy Sciences</td><td>16220</td><td></td></tr><tr><td>Human retinal endothelial cells (HREC)</td><td>Cell Systems Corp</td><td>ACBRI 181</td><td></td></tr><tr><td>MCDB131 medium</td><td>Corning</td><td>15-100-CV</td><td></td></tr><tr><td>Mouse retinal endothelial cells (mREC)</td><td>Cell Biologics</td><td>C57-6065</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;BP151-100</td><td></td></tr><tr><td>Trypsin</td><td>Gibco-Thermo Fisher Scientific</td><td>25200-056</td><td></td></tr><tr><td>&lt;strong&gt;AFM Measurement&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 &amp;micro;m Probe</td><td>Bruker</td><td>SAA-SPH-1UM</td><td>A 19 micron tall hemispherical probe with 1&lt;br/&gt; micron end radius, Spring constant 0.25N/m</td></tr><tr><td>70 nm LC probe</td><td>Bruker</td><td>PFQNM-LC-V2</td><td>A 19 micron tall hemispherical probe with 70nm end radius,&lt;br/&gt; &amp;nbsp;Spring constant 0.1N/m</td></tr><tr><td>&amp;nbsp;camera</td><td>XCAM family</td><td>Toupcam</td><td>1080P HDMI</td></tr><tr><td>Desktop to run the camera</td><td>Asus</td><td>Asus desktop</td><td>Intel i5-6600 CPU , 8GB RAM</td></tr><tr><td>Dish holder for coverslip</td><td>Cellvis</td><td>D29-14-1.5-N</td><td>29mm glass bottom dish with&lt;br/&gt; &amp;nbsp;14 mm micro-well</td></tr><tr><td>Nanowizard 4</td><td>Bruker</td><td>Nanowizard 4</td><td>Bioscience atomic force microscope mounted on an optical microscope for sensitive measurement of the mechanostructural properties (stiffness and topography) of soft biological samples</td></tr><tr><td>Phase contrast micrscope</td><td>Zeiss</td><td>Axiovert 200</td><td>Inverted microscope with 10X objective</td></tr></tbody></table>

isolation of mouse retinal capillaries and subendothelial matrix for stiffness measurement using atomic force microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

Mouse retinal capillaries 
AFM stiffness measurement of isolated retinal capillaries involves sample handling steps that could potentially damage their mechanostructural integrity. To prevent this and thereby ensure the feasibility, reliability, and reproducibility of AFM measurements, the enucleated eyes are fixed in 5% formalin overnight at 4 &#176;C prior to vessel isolation. This mild fixation protocol with reduced formalin concentration, low fixation temperature, limited fixation time, and lack...

Author Spotlight: Investigating Vascular Stiffness and Inflammation in Early Diabetic Retinopathy

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that ...

isolation-mouse-retinal-capillaries-subendothelial-matrix-for

Research

JoVE Journal

Biology

612 Views.  University of California, Los Angeles. Van vasculaire ontsteking is bekend dat het degeneratie van retinale haarvaten veroorzaakt bij vroege diabetische retinopathie, wat een belangrijke gezichtsbedreigende microvasculaire complicatie van diabetes is. Ons lab onderzoekt of en hoe mechanische signalen in de vorm van vasculaire stijfheid kunnen bijdragen aan deze abnormale retinale vasculaire veranderingen bij vroege diabetes. Er is een groeiende belangstelling voor het ontwikkelen van manieren om d...

Video: Auteur in de schijnwerpers: onderzoek naar vasculaire stijfheid en ontsteking bij vroege diabetische retinopathie

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).

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

Bioengineering

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.

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 ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

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 ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

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 ...

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 ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.
To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young&#39;s (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.
A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

We present a protocol to measure the elastic moduli of collagen-rich areas in normal and diseased liver using atomic force microscopy. The simultaneous use of polarization microscopy provides high spatial precision for localizing collagen-rich areas in the liver sections.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

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