High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition
“…The drop would have a diameter of approximately 0.5–1 cm before spin coating. Spin coat at 500 rpm for 30 secs to generate a substrate thickness of >100 μm ( Figure 1 ) ( Yoshie et al., 2019 ). Figure 1 Schematic to Illustrate the Process of Spin-Coating a PDMS Substrate Droplet onto a Dish with a Glass Coverslip to Achieve a Flattened Layer with Certain Height Be sure to align the centers of the spin-coater, the PDNS drop, and the Petri dish.…”
Summary
Cellular traction forces influence epithelial behavior, including wound healing and cell extrusion. Here, we describe a simple
in vitro
traction force microscopy (TFM) protocol using ECM protein-coated polydimethylsiloxane substrate and widefield fluorescence microscopy. We include detailed steps for analysis so readers can obtain traction forces to study the mechanobiology of epithelial cells. We also provide guidelines on when to adopt another common class of TFM protocols based on polyacrylamide hydrogels.
For complete details on the use and execution of this protocol, please refer to
Saw et al. (2017)
and
Teo et al. (2020)
.
“…The drop would have a diameter of approximately 0.5–1 cm before spin coating. Spin coat at 500 rpm for 30 secs to generate a substrate thickness of >100 μm ( Figure 1 ) ( Yoshie et al., 2019 ). Figure 1 Schematic to Illustrate the Process of Spin-Coating a PDMS Substrate Droplet onto a Dish with a Glass Coverslip to Achieve a Flattened Layer with Certain Height Be sure to align the centers of the spin-coater, the PDNS drop, and the Petri dish.…”
Summary
Cellular traction forces influence epithelial behavior, including wound healing and cell extrusion. Here, we describe a simple
in vitro
traction force microscopy (TFM) protocol using ECM protein-coated polydimethylsiloxane substrate and widefield fluorescence microscopy. We include detailed steps for analysis so readers can obtain traction forces to study the mechanobiology of epithelial cells. We also provide guidelines on when to adopt another common class of TFM protocols based on polyacrylamide hydrogels.
For complete details on the use and execution of this protocol, please refer to
Saw et al. (2017)
and
Teo et al. (2020)
.
“…Using contractile force screening (Park et al, 2015;Yoshie et al, 2018;Yoshie et al, 2019), we discovered that while the lipophilic A B C D FIGURE 1 | Pitavastatin inhibits basal-and histamine-induced airway smooth muscle (ASM) contraction. (A) As compared to no treatment (0 µM), statistically significant reductions in contraction occurred at 24 h as follows: pitavastatin (Pit) at 0.4, 2, and 10 µM and simvastatin (Sim) at 0.4 and 10 µM.…”
Despite maximal use of currently available therapies, a significant number of asthma patients continue to experience severe, and sometimes life-threatening bronchoconstriction. To fill this therapeutic gap, we examined a potential role for the 3hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor, pitavastatin. Using human airway smooth muscle (ASM) cells and murine precision-cut lung slices, we discovered that pitavastatin significantly inhibited basal-, histamine-, and methacholine (MCh)-induced ASM contraction. This occurred via reduction of myosin light chain 2 (MLC2) phosphorylation, and F-actin stress fiber density and distribution, in a mevalonate (MA)-and geranylgeranyl pyrophosphate (GGPP)-dependent manner. Pitavastatin also potentiated the ASM relaxing effect of a simulated deep breath, a beneficial effect that is notably absent with the b2-agonist, isoproterenol. Finally, pitavastatin attenuated ASM pro-inflammatory cytokine production in a GGPP-dependent manner. By targeting all three hallmark features of ASM dysfunction in asthma-contraction, failure to adequately relax in response to a deep breath, and inflammation-pitavastatin may represent a unique asthma therapeutic.
“…To measure contractile work, polydimethylsiloxane (PDMS) substrates with different stiffnesses were prepared as described previously 17,18,31 . In brief, PDMS solutions were supplied by mixing same weight ratio of component A and B of commercial PDMS (NuSil® 8100, NuSil Silicone Technologies) with different concentrations of Sylgard 184 PDMS crosslinking agent (Dimethyl, methylhydrogen siloxane, which contains methyl terminated silicon hydride units) to obtain substrates with various stiffnesses.…”
Section: Synthesis Of Compliant Silicone Substratesmentioning
confidence: 99%
“…In brief, PDMS solutions were supplied by mixing same weight ratio of component A and B of commercial PDMS (NuSil® 8100, NuSil Silicone Technologies) with different concentrations of Sylgard 184 PDMS crosslinking agent (Dimethyl, methylhydrogen siloxane, which contains methyl terminated silicon hydride units) to obtain substrates with various stiffnesses. We measured the mechanical properties of the PDMS at different crosslinker concentrations using a parallel plate rheometer (Anton Paar) and calculated the Young's moduli (Table 1) 17,18,31 . For our experiments, 50 μl of uncured PDMS was applied to .…”
Section: Synthesis Of Compliant Silicone Substratesmentioning
confidence: 99%
“…The copyright holder for this preprint (which this version posted May 16, 2020. ; https://doi.org/10.1101/2020.05.14.097006 doi: bioRxiv preprint the clean 22*22 mm (No.1) glass coverslips and cured at 100°C for two hours. For traction force microscopy, prepared PDMS substrates were coated with a layer of fiduciary particles using spin coater (WS-650 Spin Processor, Laurell Technologies) and incubated at 100 °C for an hour 17,18 . Printing on silicone substrates using UV patterning.…”
Section: Synthesis Of Compliant Silicone Substratesmentioning
6The sensing and generation of cellular forces are essential aspects of life. Traction Force 7 Microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell 8 contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, 9 their implementation remains limited in part due to various constraints, such as time-consuming 10 substrate fabrication techniques, the need to detach cells to measure null force images, followed 11 by complex imaging and analysis, and the unavailability of cells for post-processing. Here we 12 introduce a reference-free technique to measure cell contractile work in real-time, with basic 13 substrate fabrication methodologies, simple imaging, and analysis with the availability of the cells 14 for post-processing. In this technique, we confine the cells on fluorescent adhesive protein 15 micropatterns of a known area on compliant silicone substrates and use the cell deformed pattern 16 area to calculate cell contractile work. We validated this approach by comparing this Pattern-based 17 Contractility Screening (PaCS) to conventional bead-displacement TFM and show quantitative 18 agreement between the methodologies. Using this platform, we measure the contractile work of 19 highly metastatic MDA-MB-231 breast cancer cells is significantly higher than non-invasive 20 MCF-7 cells. PaCS enables the broader implementation of contractile work measurements in 21 diverse quantitative biology and biomedical applications. 22 Introduction 25Cells are not purely biochemical entities but are also subjected to physical forces and mechanics.
26Force-generation, sensing, and mechanical adaptation can be seen in nearly every aspect of our 27 physiology 1,2 . Correct recognition of responses to mechanical cues are key to health, whereas 28 dysfunctional responses are symptomatic and perhaps causative to numerous pathologies 3-6 . This 29 signifies an urgent and pressing need to quantify how cells detect and respond to mechanical 30 forces. This is a critical question in biology and biophysics, enabling new approaches in diagnosing 31 and treating diverse aspects of human health.
33Cell contractile forces are largely generated by molecular motors such as myosin, which pull the 34 filamentous actin network to perform mechanical work on the surrounding matrix 7 . There are 35 diverse methodologies to measure cell contractile work 8-10 ; however, Traction Force Microscopy 36 (TFM) has emerged as the leading approach 8-10 . TFM has revealed the roles of cell contractile 37 forces in regulating diverse physiological and pathological processes such as cell proliferation 11 , 38 differentiation 13 , migration 11,12 , nuclear polarization and deformation 15,16 , in virtually all adherent 39 cells, thus making contractile work measurements a critical aspect of quantifying biological 40 behaviors 11-16 and potentially identifying pathologies 14 .
42Although TFM is a widely useful technique, its implementation is limited in part due to 43 experimental complex...
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