Pnpp disodium salt hexahydrate

For recombinant protein capture (ELISA), 384-well plates were incubated with 2 μg ml⁻¹ of antigen overnight at 4°C. The plates were blocked for one hour with 2% non-fat dry milk supplemented with 2% goat serum. Plates were washed three times with PBS-T and primary mAbs or hybridoma cell culture supernatants were applied to wells for one hour. Plates were washed with PBS-T four times before applying 25 μl secondary alkaline phosphatase-conjugated antibody (goat anti-human IgG Fc, Meridian Life Science) at a dilution of 1:4,000 in blocking solution. After a 1 h incubation, the plates were washed five times with PBS-T and 25 μl of substrate solution (1 mg ml⁻¹ pNPP disodium salt hexahydrate, Sigma) was added to each well. The plates were incubated at room temperature for approximately 30 min before reading the optical density at 405 nm on a Biotek plate reader. Experiments with the RSV F mutants were conducted similarly.

Acid phosphatase assay was used to measure cell viability based on the conversion of 4-Nitrophenyl phosphate (pNPP) to p-nitrophenol by cytosolic acid phosphatase [45 (link)]. pNPP disodium salt hexahydrate was purchased from Sigma-Aldrich. Cells were grown in a 96 well plate at a density of 1.5 × 10⁴ cells per well and treated with increasing concentrations of 5-FU/OX (with a concentration ratio of 1:5 between OX and 5-FU) in combination with increasing concentrations of ABT-199. After 48 h treatment medium was removed and each well was washed once with 200 μL of 1× phosphate buffered saline (PBS). To each well, 100 μL of assay buffer (0.1 M sodium acetate at pH 5.0, 0.1% Triton X-100, and 7.25 mM p-nitrophenyl phosphate) was added. The plates were then incubated at 37 °C for 2 h. The reaction was finally stopped with the addition of 50 μL and colour development was assayed at 405 nm using a Clariostar plate reader (BMG Labtech, Offenburg, Germany), and applying a path length correction of 200 µL. The non-enzymatic hydrolysis of the pNPP substrate was also determined by including wells with the assay buffer and without any cells. An excel template was used to calculate the fraction affected from viability percentages and the results were analysed with the web version of Chalice Analyzer (Horizon Discovery, Waterbeach, UK) to calculate isobolograms.