Multiskan ascent spectrophotometer

Blood samples in heparinized tubes were centrifuged at 10,000 rpm for 15 min (Centrifuge 5424R, Eppendorf, Montesson, France), and plasma was collected. Plasma levels of Aβ_1-40 and Aβ_1-42 were measured using ELISA kits (Plasma Beta-Amyloid 1-40 ELISA, EuroImmun, EQ6511-9601, and Plasma Beta-Amyloid 1-42 ELISA, EuroImmun, EQ6521-9601, EuroImmun, Bussy-Saint-Martin, France) following the manufacturer’s instructions. Briefly, 25 µL of the samples were diluted in 175 µL of the dilution buffer. Then, 20 µL of biotin solution per well was incubated with 80 µL of the diluted samples, calibrators, and controls for 3 h at room temperature. The ELISA plate was washed using the washing buffer, and 100 µL per well of enzyme conjugate was added for 30 min. The wells were washed again, 100 µL per well of chromogen/substrate was added and the plate was incubated for 30 min in the dark. 100 µL of stop solution per well was added to stop the reaction, and the absorbance at 450 nm was measured with a Multiskan Ascent spectrophotometer (ThermoLab Systems, Issy-les-Moulineaux, France). The amounts of Aβ_1-40 and Aβ_1-42 in the plasma were estimated by reference to the manufacturer’s standard curve and expressed in pg/mL of plasma. A minimum of seven mice per group were used for this experiment.

Murine Il1β (559603, BD Biosciences) IL6 (555240, BD Biosciences), and TNFα (DY410, R&D Systems) were used to detect protein levels in collected media as per manufacturer’s instructions. Briefly, 96-well plates were coated overnight in capture antibodies diluted in assay diluent (1:1000) at 4°. Plates were washed thrice, then blocked in assay diluent. Wells were then filled with either recombinant standards or 100 μL media from collected samples (2 hours, room temperature. Plates were then washed 5 times after which 100 μL working detector (detection antibody + SAv-HRP reagent) was added (1 hour, room temperature). Plates were then washed 7 times and a 1:1 mixture of hydrogen peroxide and 3,3′, 5,5′ tetramethylbenzidine was added (30 minutes, room temperature). The reaction was then stopped using sulphuric acid (160 mM) and absorbance measured at 450 nm in a Multiskan Ascent spectrophotometer (Thermo Scientific)

Impact of AGuIX@Tb or ZnPy3P1 on U-251 MG cell survival was assessed by the MTT procedure, based on the measurement of mitochondrial succinate dehydrogenase activity (EC 1.3.5.1) [60 (link)]. Cells were seeded in 96 well-plates at 10⁴ cells/well and left growing for 24 h. Cells were treated with increasing concentrations of ZnPy3P1 (up to 400 µM) and AGuIX@Tb (0.1 to 2 mM) over three days. In addition, glioblastoma cells were exposed to increasing concentrations of AGuIX@Tb-P1 or AGuIX@Gd-P1 (1 to 10 µM) for 72 h. At the time, the medium was discarded and replaced by 100 µL of complete medium containing 0.5 mg mL⁻¹ MTT. The plates were incubated for 2 h at 37 °C and formazan crystals obtained were dissolved by adding 100 µL of DMSO. The plates were read at 540 nm (Multiskan Ascent spectrophotometer, Thermo Fisher Scientific, Illkirch, France). Results are expressed relative to those obtained from untreated cells (control), taken as 100. They represented quadruplicate determinations from two independent experiments (n = 8).