Cellmasktm

Experiments were performed as described in previous works. 33, 34 Briefly, HeLa cells were seeded at 8x10 5 per dish in Nunc™ Glass culture dishes. After 24 h, NPs (AuChi and AuCeO2) were added at 20 µg/mL and incubated for 24h. Nuclei (Hoechst 33342 dye, 1:20000 dilution, blue) (Life Technologies, Madrid, Spain) and cell membranes (CellMaskTM, 1:10000 dilution, green) (Life Technologies, Madrid, Spain) were stained following manufacturer's protocol. 1, 2 Fluorescence images were obtained in vivo with an Olympus (FV1000-spectral) confocal microscope. 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), and 488 nm (exciting CellMask, green) recording the emission from 415 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.
Z-stack study across the depth of the cells was recorded with an interslice distance of 400nm.
Fiji ImageJ program was used to analyse the images.

We used coverslips coated with poly-L-lysine to seat the HEK293T cell, and images were taken 48h after transfection and before previous incubation with the plasma membrane marker CellMask TM (Invitrogen C10046). A Leica TCS SP5 II inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63×/1.40 NA oil immersion objective was used to acquire the images. All images were obtained from living cells in phosphate-buffered saline at room temperature (22-24°C), digitalized with a resolution of 1024×1024 pixels, 200 Hz velocity, and 6-line average in sequential scanning mode. To minimize crosstalk between channels, eGPF was excited with a 488-nm Argon laser simultaneously with CellMaskTM at 633 nm during the scanning process. For live imaging, mCherry was excited sequentially using a 594-nm He-Ne laser between scan lines. The emitted signals were then collected from the three distinct channels, with the following bandpass lter settings: 492 to 550 nm for eGFP detection, 598 to 630 nm for mCherry, and 640 to 700 nm for CellMaskTM. We used the ImageJ 1.44p software (Image J v.1.53c, Wayne Rasband, National Institutes of Health, USA) (Schneider et al., 2012) (link) to process the images. Colocalization analyses were performed by calculating Manader's colocalization coe cient using the JACoP plugin (Bolte & Cordelieres, 2006) (link).

The cellular uptake experiment was performed using confocal fluorescence microscopy on living cells. Cells were seeded (10 6 cells•cm -2 ) on culture dishes with a glass bottom (Fluorodish from World Precision Instruments, Stevenage, UK). After seeding, cells were incubated with 50 µg.mL -1 of (E) or (Z) isomers for 24 h. Cells were also loaded 15 min with CellMask TM (Invitrogen, Cergy Pontoise, France) at 5 µg•mL -1 , for membrane staining. Then, cells were washed twice with culture medium and fluorescence imaging was performed on living cells with a LSM780 LIVE confocal microscope (Carl Zeiss, Le Pecq, France), at 750 nm for isomers detection and 561 nm for cell membranes. All images were obtained with a high magnification (Plan-Apochromat 40x/1.3 Oil DIC).