Safire microplate reader

SEAP activity (∆A₄₁₀/min) was determined as previously described^{31 (link)}. Briefly, culture supernatants were diluted with water as required (typically 1/100 to 1/1000), and 180 μl was transferred to a 96-well plate. The enzymatic reaction was initiated when 20 μl of SEAP assay solution (20 mM p-nitrophenyl phosphate; pNPP, 1 mM MgCl₂ and 1 M diethanolamine pH 9.8) were added, and absorbance was read at 410 nm in 1 min intervals at room temperature to determine the pNPP hydrolysis rates (Safire microplate reader, Tecan, Austria). Data are expressed as the mean of one experiment performed in triplicate with error bars representing standard deviations. Each sample was independently assayed for SEAP activity three times to minimize pipetting errors.

Cell cytotoxicity was evaluated using CellTiter 96 cell proliferation assay (Promega, Madison, WI, USA). Cells (5 × 10⁴ /well) were treated with free Dox, AS1411, control DNA-Dox adduct, and AS1411-Dox adduct (respectively) in FBS-free medium. All treatments were suspended in PBS. After incubation for 1 h (37°C, 5% CO₂), medium was removed, and fresh medium (10% FBS, 100 IU/mL penicillin-streptomycin, 200 μL) was added for further cell growth (48 h). Then medium was removed again, and CellTiter reagent (20 μL) diluted in fresh medium (100 μL) was added to each well and incubated for 1–2 h. The absorbance (490 nm) was recorded using a microplate reader (Tecan Safire microplate reader, AG, Switzerland). Cell viability was determined according to the manufacturer’s description.

Female homozygous athymic nude NCr mice (N=24) bearing 4T1 tumours grown over 7 days were injected with a mixed dose containing Doxil (10 mg doxorubicin per kg body weight) and ⁸⁹Zr-NRep (20.3±3.9 μCi, Supplementary Table 2). At predetermined time points (6, 24 and 48 h), animals were killed and perfused with PBS. Tumours were collected and weighed. Larger tumours were divided into portions of ∼50 mg. The resulting tumour samples were counted using a Wizard2 2480 Automatic Gamma Counter (Perkin Elmer, Waltham, MA). The doxorubicin concentration was quantified as previously reported26 (link). Briefly, immediately after gamma counting, tumour samples were homogenized in lysis buffer (10:1 v/w ratio) using a hand-held electrical homogenizer. Aliquots of 200 μl of homogenate were transferred to a new tube, and water (200 μl), Triton X-100 (10 % solution in water, 100 μl) and finally acidified isopropanol (0.75 N HCl, 1.5 ml) were added. The mixture was vortex mixed and left at −20 °C for 16 h. Samples were then vortexed, centrifuged at 15,000 r.p.m. for 20 min, and aliquots of 200 μl were measured on a 96-well plate using a Safire microplate reader (Tecan, Männedorf, Switzerland). A calibration curve was generated by adding increasing amounts of doxorubicin to tumour sample homogenates (prepared as described above) from animals not treated with Doxil.

The biofilm of WT, Δhfq, and hfq complementing strains of P. ananatis was quantified as previously described by Santander and Biosca (2017) (link) with slight modifications. An aliquot of 160 μl broth culture diluted to an OD_600nm of 0.5 in half-strength LB [0.5% (w/v) NaCl, 0.5% (w/v) tryptone, and 0.25% (w/v) yeast extract; pH 7.2] was made into each well of a polystyrene 96-well microplate (Nunc^TM MicroWell^TM, Thermo Scientific, Waltham, MA, United States) and incubated for 24 h under static conditions. Eight replicates per P. ananatis strain were included in each experiment with sterile half-strength LB broth serving as a negative control. Thereafter, the inoculated 96-well plates were inverted to remove the excess LB broth, air-dried, and incubated at 60°C for 40 min to heat-fix the biofilms. The biofilms were stained with 1% crystal violet (220 μl) for 15 min before being rinsed with distilled water. After rinsing and invert-air-drying the microplate, 220 μl of ethanol:acetone in 8:2 ratio was added to the wells to solubilize the crystal violet dye for 20 min at room temperature. The solubilized biofilm was measured at OD₆₀₀ using Safire Microplate Reader (Tecan, Research Triangle Park, NC, United States), and this assay was repeated three times.

The ability of A. brasiliensis to scavenge DPPH free radicals was assessed using the previously described decolorization method, with minor modifications [16 (link), 17 (link)]. Briefly, powdered mushrooms were extracted with 50% methanol (50 mg/mL) at 60°C for 60 min. Trolox dissolved in 50% methanol was used as a standard antioxidant. Diluted extracts (20 μL) were combined with 200 μL of 150 μmol/mL DPPH in 50% methanol. All samples were prepared in triplicate. Following incubation in the dark at room temperature for 30 min, the absorbance at 517 nm was read using a Safire microplate reader (Tecan, Salzburg, Austria). The effect of natural color changes of the samples was controlled for by using a blank, and the data was expressed as equivalents of Trolox.