Fv1000 spectral confocal microscope

HeLa cells were seeded at 8 × 10⁵ per dish in Nunc™ Glass culture dishes (Thermo Fisher Scientific, Sant Cugat del Vallés, Spain). After 24 h, different NPs (AuCeO₂ or TPP–AuCeO₂) at 20 µg/mL were added and incubated for 24 h (37 °C, 5% CO₂ atmosphere (Linde, Barcelona, Spain)). Nuclei (Hoechst 33342 dye, 1:20,000, blue) (Life Technologies, Madrid, Spain), cell membrane (CellMaskTM, 1:10,000, green) (Life Technologies, Madrid, Spain), and mitochondria (MitoTracker™, 1:2,000, red) (Thermo Fisher Scientific, Sant Cugat del Vallés, Spain) were stained following the manufacturer’s protocol [39 (link),40 (link)]. Fluorescence images were obtained in vivo using a FV1000-spectral confocal microscope (Olympus, Hamburg, Germany). Samples were maintained at 37 °C under 5% CO₂ atmosphere during imaging and were illuminated simultaneously with laser light at 405 nm (exciting Hoechst, blue), 488 nm (exciting CellMask, green), and 561 nm (exciting MitoTracker, red), recording the emission from 425 to 603 nm in separate channels. The reflection signal of NPs was split by using a dichroic mirror (20/40) after irradiating at 633 nm. A Z-Stack study across the depth of the cells was performed with an interslice distance of 400 nm [41 (link),42 (link)]. The Fiji image analysis software was used to analyze the images [43 (link)].

Cells were grown on glass coverslips in 6 well plates (Fisher), fixed with 4% paraformaldehyde, permeabilized with 0.1% triton X, and blocked in 1% BSA. Slides were washed and coverslips mounted using Fluoro-Gel II with Dapi (Electron Microscope Sciences). Images were acquired at room temperature with an Olympus FV1000 spectral confocal microscope, a UPLFLN 100X objective lens, NA 1.30, and with Olympus FLOWVIEW acquisition software.

Young, H₂O₂-treated, and aged oocytes were treated with two fluorescent dyes to visualize natural fatty acids and plasma membrane [40 (link)]. To stain oocyte plasma membranes, CellMask Deep Red Plasma Membrane Stain (C10046, Invitrogen, Grand Island, NY, USA) was used at 2.5 μg/mL in media for 30 min. BODIPY fatty acid 500/510 (D-3823, Invitrogen) was used for staining natural fatty acids in mouse oocytes. These oocytes were washed three times with media and transferred to a glass-bottom confocal dish (SPL Life Sciences, Pocheon, Korea). Live-cell images were captured with an Olympus FV1000 spectral confocal microscope (Olympus, Tokyo, Japan) equipped with a warm plate or with an LSM 710 confocal microscope (Carl Ziess, Oberkochen, Germany).

To perform a mechanical stimulus we used a home made equibiaxial stretching device adapted from Quaglino et al.^{20 (link)} for live imaging in an Olympus FV1000 spectral confocal microscope (Olympus Co., Japan). The device applies controlled equibiaxial strain to cells attached to flexible membranes, by pulling a rigid plastic ring that forms the walls of the cell culture chamber, over the clamped membrane. More details on the principle of stretching based on indenter designs are described in^{51 (link)–53 (link)}. The stretching device consists in an aluminum screw-top support that contains: a two cylindrical pieces membrane holder (Delrin® made) where the silicone membrane (40 mm diameter) is clamped; a Teflon® indenter ring (30 mm diameter) that forms the walls of the cell culture chamber; and a Delrin® made flange that pushes the indenter ring that stretches the silicone membrane when the aluminum screw-top is turned down. The degree of stretching can be characterized in terms of thread turns of the aluminum screw-top.