Probing TGF-β1-induced cytoskeletal rearrangement by fluorescent-labeled silica nanoparticle uptake assay

Abstract

Cytoskeletal proteins are essential in maintaining cell morphology, proliferation, and viability as well as internalizing molecules in phagocytic and non-phagocytic cells. Orderly aligned cytoskeletons are disturbed by a range of biological processes, such as the epithelial–mesenchymal transition, which is observed in cancer metastasis. Although many biological methods have been developed to detect cytoskeletal rearrangement, simple and quantitative in vitro approaches are still in great demand. Herein, we applied a flow cytometry-based nanoparticle uptake assay to measure the degree of cytoskeletal rearrangement induced by transforming growth factor β1 (TGF-β1). For the assay, silica nanoparticles, selected for their high biocompatibility, were fluorescent-labeled to facilitate quantification with flow cytometry. Human keratinocyte HaCaT cells were treated with different concentrations of TGF-β1 and then exposed to FITC-labeled silica nanoparticles. Increasing concentrations of TGF-β1 induced gradual changes in cytoskeletal rearrangement, as confirmed by conventional assays. The level of nanoparticle uptake increased by TGF-β1 treatment in a dose-dependent manner, indicating that our nanoparticle uptake assay can be used as a quick and non-destructive approach to measure cytoskeletal rearrangement.

Platelets Regulates Cell Viability and VEGF-A mRNA Expression in HaCaT Cell Line

“…The cell morphology was analyzed by fluorescence using dihydrochloric salt, 4',6diamidino-2-phenylindole (DAPI -Molecular Probes Inc., USA) to stain the nucleus and phalloidin green to stain the cytoskeleton in cells cultured in 12-well plates. After coculture and washing, 500 µl/well of 4% paraformaldehyde (PFA, Vetec Química/ Sigma-Aldrich, Inc.) was added for 10 minutes at room temperature (RT) [18]. Cells from 8 wells were washed twice with D-PBS-CM.…”