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High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition
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Summary June 1st, 2019
We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-β dependent increase in contractility during the epithelial-to-mesenchymal transition.
Transcript
My lab is interested in mechanobiology, which examines how cells exert and are influenced by mechanical forces.One way we can quantify mechanobiology is measuring cell contractility.In adherent cells, contractility manifests in traction forces exerted on the cell substrate.We quantify these forces using a methodology generally referred to as traction force microscopy or TFM.And TFM's implemented in a broad variety of different methods, but a common aspect is that it is a rather serial and slow lab technique done typically in a single dish like this one here.This restricts us to evaluating one population or condition at a time.Throughout in cell culture was improved some time ago but creating multiwell dishes and we thought, wouldn't it be great it we could to TFM with the same increased throughput and the same efficiency of time and reagents?And this was our solution.So we created a 96 well traction force dish.And it looks very similar to a conventional 96 well tissue culture dish, however with this dish we can not only examine 96 different cell culture conditions, but also quantify their cell contractility.This presents a much higher throughput solution than single dish approaches, allowing us to examine diverse cell conditions and contractile responses.During cancer metastasis, cancer cells travel from their primary site to secondary sites, spreading the disease in our body.This metastatic behavior of cancer cells accounts for 90%of cancer-related death.One of the proposed mechanisms for epithelial cancer cells to acquire a more migratory and invasive phenotype is epithelial-mesenchymal transition, EMT.During EMT, they become more migratory and invasive, changing their physical behavior.To better understand these changes in physical behavior, we utilize our soft silicon substrate to measure changes in contractility of mouse mammary gland cells with EMT induction by TGF-In this study, we investigated how different concentrations and incubation time of TGF-affect cells'physical behavior.Polydimethylsiloxane is a very compliant rubber with the lowest Young's modulus of about 0.6 kilopascal.The stiffness of this material also can be tunable using different concentrations of crosslink agent including SYLGARD 184.To measure mechanical properties of PDMS we use a rheometer.After mixing part A and part B of the PDMS, in a one to one weight ratio and adding the curing agent with the specified weight percentage, load the prepared batch on the rheometer plate.Make sure to load enough of the sample to completely cover the spindle area.It's important to make sure the spindle touches the sample completely with no free space left at its edge.Any extra sample should be carefully trimmed and wiped from the plate.Measure the final storage modulus and time dependency of each sample's viscoelasticity by doing time and frequency sweeps at an oscillatory shear strain of 0.5%Newsha]This graph is one example of time seq test, having done to measure rheological behavior of polydimethylsiloxane with applied oscillatory shear strain of 0.5%and temperature of 100
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