96 well pcr plate

TSA assays were performed in 96-well PCR plates (VWR) using SFX96 Real-Time PCR reactor (BioRad). The protein stability was assayed based on the increased fluorescence of the dye upon binding to the hydrophobic core of the unfolded protein. SYPRO Orange dye (5000 stock, Invitrogen) was added to the protein sample at 2 mg ml^-1 in a 1:500 ratio. The protein samples were assayed against various buffer systems (pH gradient) as well as buffer components [30 (link), 31 (link)]. The volume of each sample was 50 μL, the final protein concentration in each assay was 0.2 mg ml^-1 (5,7 μM). Melting curve (in terms of increased fluorescence) of each sample was plotted against the temperature gradient (293–373 K) and the temperatures of the inflection points (T_m’s) were used as indicators of the thermal stability of each sample.

Stock solutions for platinum-based agents (10 mM cisplatin and carboplatin) and a proteasome inhibitor (1 mM bortezomib) were prepared using DMSO (Sigma-Aldrich; stored at −80 °C), further diluted in 1xPBS to the appropriate concentration, and plated in 96-well PCR plates (VWR; stored at −20 °C). The pharmaceutical compounds were screened at nine concentrations (2–1024 µM cisplatin/carboplatin and 1–10,000 nM bortezomib) using a 2-fold dilution series with matched DMSO concentration vehicle controls. The pharmaceutical compounds were at room temperature (18-25 °C) when added to cells. Proteasome activity was assessed using the Proteasome-Glo Chymotrypsin-like assay (Promega) with bortezomib-treated cells seeded in 96-well clear, flat-bottom microplates (Corning Life Sciences) at a density of 7.5 × 10³ cells per well in 100 µl culture medium (RPMI or DMEM basal medium supplemented with 5%, 10% or 15% FBS or without FBS, and HuMEC Basal Serum-Free medium supplemented with epidermal growth factor, hydrocortisone, isoproterenol, transferrin, insulin, and bovine pituitary extract (Life Technologies)).