Dulbecco s phosphate buffer saline dpbs

Cells were seeded onto round glass coverslips of 10 mm diameter previously coated with fibronectin-gelatin (2 μg/cm²). Confluent cells were fixed with 3% paraformaldehyde (PFA) for 15 min in the dark. In order to perform quenching, blocking and permeabilization, cells were then incubated with a solution of 1% bovine serum albumin (BSA), 0.1 % Triton X-100 and 50 mM glycine in Dulbecco’s Phosphate Saline Buffer (dPBS, Sigma Aldrich) for 30 min at room temperature (RT). Primary antibodies diluted in dPBS-2% BSA and 0.7 mM CaCl₂ and 0.7 mM MgCl₂⁺ were added for 1 h at RT; secondary antibodies were incubated for 30 min at RT. Nuclei were stained with DAPI. Glass slides were mounted with a fluorescence mounting medium (Dako), and images were acquired with a Leica DM3000 microscope (Leica, Wetzlar, Germany) using a Leica DFC320 digital camera (Leica).

The murine embryonal carcinoma cells line P19 (ATCC, Manasass, VA, USA, CRL-1825) was maintained in Minimum Essential Medium Alpha Modification (α-MEM; Sigma-Aldrich, Vienna, Austria) supplemented with 7.5% newborn calf serum (Sigma-Aldrich, Vienna, Austria), 2.5% fetal bovine serum (Sigma-Aldrich, Vienna, Austria), and 1% antibiotic/antimycotic solution (Sigma-Aldrich, Austria) under standard cell culture conditions at 37 °C in a 5% CO₂ humidified atmosphere. The medium was changed every three days, and cells were passaged at 90% confluency. After a washing step with Dulbecco’s Phosphate-Saline Buffer (DPBS; Sigma-Aldrich, Vienna, Austria), cells were enzymatically detached with 3 mL 0.25% Trypsin-EDTA (Sigma-Aldrich, Vienna, Austria) and subsequently pelleted at 140 × g for five minutes. The supernatant was aspirated, and the cell suspension was adjusted to required cell densities with a complete α-MEM medium. P19 cells were passaged twice or three times per week at a ratio of 1:4–1:6.

U-251 MG cells were seeded in 6 well-plates at 10⁵ cells/well and left to grow over 48 h. Cells were treated with increasing concentrations of nanoparticles (0.5 to 5.0 µM AGuIX@Tb-P1 or AGuIX@Gd-P1) over 48 h. After incubation, cell layers were washed with 2 mL of Dulbecco’s Phosphate buffer saline (DPBS, Merck-Sigma) and cells were suspended with 0.5 mL of 0.05% (w/v) Trypsin/0.02% (w/v) EDTA solution (Invitrogen) per well for 5 min at 37 °C. Complete medium (0.5 mL RPMI containing 10% (v/v) fetal calf serum) was then added. The cell suspension obtained was centrifuged at 1000 g for 5 min at 4 °C. Cell pellets were washed with 1 mL of DPBS and centrifuged again. Cells were suspended finally in 0.5 mL of DPBS and left on ice. Fluorescence of P1 was measured in 5000 cells/sample by flow cytometry (Gallios Analyzer, Beckman Coulter France, Roissy, France), with excitation/emission settings at 638 nm and 660/30 nm. Results obtained from nanoparticle uptake were expressed relative to those obtained reaching the maximum of nanoparticle absorption taken as 100. Results are expressed as mean ± SD from triplicate determinations from 3 independent experiments.