Wallac wizard 3 1480

Co²⁺ uptake by the SnO₂ and SnO₂/SiO₂ fibers calcined at different temperatures and with different heating rates was studied at a pH of 6. 20 mg of ground fibers was weighed into 20 mL scintillation vials and 10 mL of 0.01 M NaNO₃ solution containing 30 Bq mL^{−1 57}Co²⁺ was added into the vials. The pH of the solution was adjusted to 6 with a small volume of NaOH. The samples were equilibrated in a constant rotary mixer (50 rpm) for 24 hours after which the equilibrium pH was measured. The samples were phase separated by centrifugation at 4000 rpm (2100g) and syringe filtration (Acrodisc LC PVDF, 0.2 μm). The ⁵⁷Co²⁺ uptake efficiency of each sample was determined by pipetting 5 mL of the filtrate into a scintillation vial and measuring the remaining activity with a PerkinElmer Wallac Wizard 3′′ 1480 automatic gamma counter. The ⁵⁷Co²⁺ uptake results are presented by means of distribution coefficient K_d, that describes the distribution of the adsorbate between the adsorbent and solution: where c₀ (Bq L⁻¹) is the initial concentration, c_eq (Bq L⁻¹) is the equilibrium concentration, V (mL) is the volume of the solution and m (g) is the mass of dry adsorbent. Background activity was subtracted before the calculations. Uncertainty of K_d was calculated using the error propagation law.

HT29 cells (25000 cells per well) were seeded in a 24 multi-well dish in complete medium. After 24 hours, the supernatant was removed and the cells were washed gently with PBS. In each well, 300 μL of the competition media (25 mM HEPES pH = 7, containing 0.2% BSA, completed with MEM) containing 1.85.10⁻³ MBq of [¹¹¹In]DOTA-Ts29.2 were added. For competition, Ts29.2 or DOTA-Ts29.2 from 0 to 1.10⁻⁷ M was included in the reaction media. After 1 hour of incubation under gentle shaking, the supernatant was removed and each well was washed gently twice with PBS containing 2% of BSA. The radioactivity was recovered using 400 μL of an aqueous 1 N NaOH solution. The radioactivity in pooled supernatants and washings as well as in cell fractions was measured using a γ-counter (Wallac 1480 Wizard® 3‘‘, Perkin Elmer). The percentage of binding was calculated taking into account the value obtained in the absence of non-radioactive antibody.

Strains were grown overnight in the presence of ³H-labeled thymidine (Perkin Elmer). Cultures were then incubated in a Maxisorp 96-well plate coated with either porcine type II mucus (Sigma-Aldrich) or human mucus, followed by extensive washing with PBS (Oxoid) and lysis of the remaining adherent cells. Sample radioactivity was measured using a Wallac 1480 Wizard 3 automatic gamma counter (Perkin Elmer). Full experimental details are described in Vesterlund et al. (2005) (link). Each assay was performed in three biological repeats, with each a minimum of three technical replicates per strain.

The immunoreactivity of each ⁶⁴Cu-labelled immunoconjugate was determined by a conventional saturation assay according to the method recommended by Denoël et al. [28 (link)]. Briefly, increasing concentrations of BT474 cells (1.10⁶, 2.10⁶, 5.10⁶ and 10.10⁶ cells per tube) were incubated in 0.5 mL of binding media (25 mM HEPES pH 7, containing 0.2% of BSA, completed with Dulbecco’s Modified Eagle’s Medium (DMEM) F12 (1/1, v/v) glutamax) with 0.60 pmol of the appropriate immunoconjugate (6.8–27.4 GBq/µmol) in a final volume of 1 mL. After 30 min of gentle shaking, the samples were centrifuged at 460× g for 8 min at 4 °C. The supernatants were removed, the cell pellets were washed with PBS containing 0.2% BSA and centrifuged again at 460× g for 8 min. The supernatants and pellets collected were recovered separately for radioactivity counting using a γ-counter (Wallac 1480 Wizard^® 3‘‘, Perkin Elmer, Villebon sur Yvette, Every, France). The IRF was determined by performing a rectangular hyperbolic fit (one site-specific binding, GraphPad Prism 9.4.1) of the binding curve obtained by plotting B/(B+S) as a function of cell concentration, where B and S are the pellet and supernatant activities counted, respectively. IRF was obtained by extrapolating the quadratic hyperbola value at infinite antigen concentration. All experiments were performed in triplicate.