Mouse il 2 duoset elisa kit

To measure IL‐2 release, we used the Mouse IL‐2 DuoSet ELISA kit (R&D Systems; DY402‐05) following the manufacturer's protocol. First, 10⁶ cells in 150 μl medium per well were seeded into a 48‐well plate and allowed to settle for 30 min. Cells were then stimulated with IL‐1β (Peprotech, cat. No. 211‐11B) in 50 μl medium per well at a final concentration of 10 ng/10⁶ cells. For unstimulated controls, 50 μl medium only was added. After 24 h, plates were centrifuged (300 g for 5 min), and supernatants were transferred to a new plate. Supernatants were stored at −80°C until IL‐2‐ELISA analysis. Absorbance readings were acquired on a VersaMax Microplate Reader (Molecular Devices) at 450 nm. IL‐2 release was assayed on three independent days in triplicate. The obtained results were normalized based on the EL4 WT IL‐2 release (Appendix Fig S2C).

To measure IL2 release, we used the Mouse IL-2 DuoSet ELISA kit (R&D Systems; DY402-05) following the manufacturer’s protocol. Briefly, 10⁶ cells in 150 µl medium per well were seeded into a 48-well plate. Cells were allowed to settle for 30 min before being stimulated with IL1β in 50 µl medium per well at a final concentration of 10 ng/ml. For the unstimulated control, 50 µl medium was added. After 24 h, plates were centrifuged (300 g for 5 min), and supernatants were transferred into a new plate. Supernatants were stored at −80°C until IL2-ELISA analysis. Absorbance readings were acquired on a VersaMax Microplate Reader (Molecular Devices) at 450 nm. IL2 release was assayed on 3 independent days in triplicate. The obtained results were normalized based on the EL4 WT IL2 release.