The cell line HeLa RFP-FUSP525L has been generated and maintained as described in supplemental material. For sample preparation, 50,000 cells were seeded in each μ-Dish 35 mm high Grid-50 Glass Bottom (ibidi) in 2 mL of culture medium. Where specified, 200 ng/mL doxycycline (Sigma-Aldrich) were added to the medium for RFP-FUSP525L induction. The next day, cells were treated with 0.5 mM sodium arsenite (Sigma-Aldrich) for 90 min, or left untreated, and then prepared as follows: washed with PBS (Sigma-Aldrich), fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, incubated 5 min with PBS containing 0.1 M glycine, washed two times with PBS and left in PBS for Brillouin acquisition.
μ dish 35 mm high grid 50 glass bottom
Manufactured by Ibidi
The μ-Dish 35 mm high Grid-50 Glass Bottom is a laboratory equipment product designed for cell culture applications. It features a 35 mm diameter culture dish with a high sidewall and a grid-patterned glass bottom. The grid pattern provides a visual reference for locating and positioning cells within the dish.
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2 protocols using μ dish 35 mm high grid 50 glass bottom
1
Brillouin Imaging of Arsenite-Stressed Cells
2
Investigating Cytoplasmic Stiffness in HeLa Cells
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HeLa cells (50,000) were seeded in each μ-Dish 35 mm high Grid-50 Glass Bottom (Ibidi) in 2 mL of culture medium. Doxycycline (200 ng/ml; Sigma-Aldrich) was added to the medium for ectopic protein induction. The next day, cells were prepared as follows: washed with PBS (Sigma-Aldrich), fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed with PBS, incubated 5 minutes with PBS containing 0.1M glycine, washed 2 times with PBS and left in PBS for confocal and Brillouin acquisition, as previously described (Antonacci et al., 2018 (link)). For the analysis shown in Figure 3 E, a mask on portions of the cytoplasm showing high fluorescent signal was first created to localize different subcellular regions across the associated Brillouin images, in turn providing a measure of the frequency shift (a stiffness indicator).
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