2.4. Solubility Assay

The determination of the aqueous solubility of compounds in this assay is based on the principle of turbidimetry. Turbidimetric methods rely on the measurement of light scattering from precipitate in solution to determine the solubility. Precipitation is identified by an absorbance increase due to blockage of the light by the particles at the wavelength of 570 nm. Compounds, stored in matrix vials (Thermo Fisher) at a stock concentration of 30 mM or 10 mM in 100% Dimethyl sulfoxide (DMSO), were used to make a serial dilution (dose-response) in a 96-well v-bottom propylene plate (Greiner, 651201). Serial dilutions are made to perform the solubility assay; they were made row-wise and start with undiluted compound (30 mM or 10 mM) in the first well and were then 1 over 3 diluted further on. The serial dilutions were eight doses long and contain six compounds per plate maximal (rows B-G). Columns 1 and 12 were filled with 100% DMSO for control purposes. The dose-response plates were 200-times diluted in 300 µL Phosphate Buffered Saline (PBS; pH = 7–7.2 without Ca²⁺/Mg²⁺) (Gibco, 14190-144) by transferring 1.5 µL of serially diluted compound. This results in final starting concentration of 150 µM (starting from 30 mM) or 50 µM (starting from 10 mM) at 0.5% DMSO. Dilutions were made by diluting one 96-well plate in two 96-well, flat-bottom, polystyrene plates (Greiner, 655101). These plates were then incubated at room temperature for 1h. After 1h incubation, the plates were read on the envision (Perkin Elmer) at 570 nm.

The data of this assay is reported as “Soluble at” value for each compound. This “soluble at” value represents the concentration where the compound is still soluble and is the concentration before the first precipitated concentration. The first precipitated concentration is the concentration where the absorbance value is more than five standard deviations higher than the average background absorbance. The average background absorbance and the standard deviation are calculated on the 0.5% DMSO controls in columns 1 and 12.