Infinite m1000 pro reader

The enzyme-coupled ATPase assay uses two separate fast enzymatic reactions to couple ATP regeneration to NADH oxidation. The typical reaction contained 100 nM protein in buffer containing 50 mM potassium acetate, 20 mM KOH-HEPES pH 7, 5 mM magnesium acetate, 5% glycerol (v/v), 0.2 mg ml⁻¹ BSA, 3 mM phosphoenolpyruvate (PEP), 0.3 mM NADH and excess pyruvate kinase and lactate dehydrogenase enzyme mix (Sigma). The reaction mixture was incubated for 10 min at 30 °C and the reaction was started by addition of ATP (1.5 mM final). The rate of ATP hydrolysis was monitored by measuring a decrease in the absorption at 340 nm using the Infinite M1000Pro reader (Tecan). Resulting curves were fit to a linear model using GraphPad Prism version 9.

Effect of hypoculoside on the growth of three bacterial strains Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 9027) and Klebsiella aerogenes (ATCC 13048) was assessed as previously described^{43 (link)}. Cytotoxicity of hypoculoside on the A549 human lung cell line and the pancreatic carcinoma cell lines MIA PaCa-2 and PANC-1 was determined as described before^{43 (link)}. A549 and MIA PaCa-2 were seeded at 1,500 cells per well, and PANC-1 cells were seeded at 2,500 cells per well in a 384-well microplate. Cells were treated with hypoculoside at various concentrations and incubated for 72 hours at 37 °C in the presence of 5% CO₂. Cytotoxic effect of hypoculoside was measured using the PrestoBlue™ cell viability reagent (Life Technologies). Following incubation of the microplates with the dye for 2 hours, the fluorescence reading (Excitation/Emission: 560 nm/590 nm) was recorded using the Tecan Infinite M1000 Pro reader.

HEK293 cells stably harboring plasmids for Tet-inducible expression of tBRCT domains were seeded at a density of 60,000 cells/35mm dish. Compound addition or inducible expression of BRCA1 tBRCT were performed as indicated, when cells were ∼30% confluent, before exposure to 1Gy irradiation. Cells were replenished with media every 3^rd day for 7 days, re-suspended in cell dissociation buffer, which was neutralized in 1x PBS. Cells were mixed thoroughly to ensure single cell suspension before measurement of viability using Calcein AM dye and/or cell counting with a hemocytometer. Cell survival fractions were measured using fluorescence readout of Calcein AM with the Tecan Infinite M1000 Pro reader (485nm excitation and 515nm emission) and data were normalized to untreated controls across treatment groups.

The cytotoxic effect of the isolated compounds on the A549 human lung carcinoma cell line was determined as described previously [92 (link)]. A549 cells at a concentration of 3.3 × 10⁴ cells/mL in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Life Technologies, Bleiswijk, The Netherlands) were treated with isolated compounds at concentrations ranging from 0.1 μM to 200 μM and incubated for 72 h at 37 °C in 5% CO₂ and 95% relative humidity. Cytotoxic effect of the compounds was measured after the addition of PrestoBlue™ cell viability reagent (Thermo Fisher Scientific, USA) and incubation for 2 h by reading of the plates’ fluorescence at an excitation wavelength of 560 nm and emission wavelength of 590 nm using the Tecan Infinite M1000 Pro reader. Puromycin served as the standard control.

A549 and HepG2 cells were seeded at 1500 and 2500 cells per well, respectively, in 384-well microplates and incubated at 37 °C in the presence of 5% CO₂. The next day, cells were treated with compounds then incubated for an additional 72 h. To assess the cytotoxic effect of the compounds PrestoBlue cell viability reagent (Life Technologies, Carlsbad, CA, USA) was used. After the 72 h incubation with compounds, the 384-well assay plates were incubated with this viability indicator dye for 2 h (A549) and 4 h (HepG2) before fluorescence reading at Ex/Em = 560 nm/590 nm on the Tecan Infinite M1000 Pro reader. Puromycin (data not shown) was used as the positive control against both cell lines. All tests were executed in triplicate on two separate experiments.

For assays in 2D culture, cells were seeded at predetermined optimal densities in 96-flat-well plates (Corning). For assays in 3D cultures, 1000 cells per well were seeded in Poly-HEMA-coated (0.6 mg per well) 96 round-well plates (Corning). In addition, cells were pelleted at 300 g for 3 min to facilitate more homogenous spheroid growth. Twenty-four hours post seed, compounds for treatments were added and incubated for another 96 h. XTT assay reagent (neoFroxx) was added, and after development, absorbance was read at 463 vs. 670 nm on a TECAN Infinite M1000 Pro reader. Data shown are expressed as percentage of metabolic activity compared to untreated or vehicle-treated cells, corrected by subtraction of blank values. Titration curves were fitted with a “log(inhibitor) vs. response—variable slope (four parameters)” fit (GraphPad Prism). Data were weighted by 1/Y² and the Hill slopes were constrained to >“−3”. Antiproliferative effects were compared by calculating the area under the curve with a 95% confidence interval. Nonoverlapping areas were considered different. Single concentration points were compared by the two-sided Student’s t test or two-sided ANOVA where applicable and corrected for multiple testing where due; see individual figure captions for detailed description of applied statistical tests.