Mouse pai 1 duoset elisa

The levels of VEGF-A (vascular endothelial growth factor A) and PAI-1 (plasminogen activator inhibitor 1) were measured in conditioned media from Pten/WT and Pten/KO B16 F10 and Renca cells using commercially available enzyme-linked immunosorbent assays Mouse VEGF DuoSet ELISA and Mouse PAI-1 DuoSet ELISA (both R&D Systems, USA), according to the manufacturer’s protocol. Concentrations were calculated against the standard curve using recombinant proteins provided in the kits. Absorbance (450 nm) was measured using a VarioScan Lux (Thermo Fisher Scientific, Waltham, MA, USA).

To assess Pten/WT and Pten/KO cells’ susceptibility to cisplatin treatment in hypoxia (1% pO₂), cells were seeded in 96-well plates under standard oxygen conditions and allowed to adhere to the culture surface. After 24 h, the medium was changed to a pre-equilibrated hypoxic medium, and the cells were cultured in an XVivo X3 workstation (Biospherix, USA) in 1% pO₂ and 5% CO₂ at 37 °C for a further 24 h. Next, cisplatin was added at the final testing concentrations, as previously used in normoxic conditions, and Alamar Blue was used, as described above. The conditioned media were collected from cells cultured in hypoxia for 72 h without drugs, and PAI-1 secretion was detected using a Mouse PAI-1 DuoSet ELISA (R&D Systems, USA), as described above.