Human il 2 elisa kit

For Jurkat cell stimulation assay shown in Fig 2E and F, Appendix Fig S1C and Fig 5C, Jurkat‐PD‐1 cell transfected with NFAT‐luciferase reporter (designated JP‐luc) was generated. Raji stably expressing hPD‐L1 was generated (designated Raji‐L1) as antigen‐presenting cells (APC). Raji cells or Raji‐L1 cells were preincubated with 1 μg/ml of SEE superantigen (Toxin Technology, ET404) in serum‐free RPMI medium for 1 h at 37°C. JP‐luc cells were pretreated with DMSO, MB, or aPD1 antibody for 1 h before mixing with SEE‐loaded Raji or Raji‐L1 cells at ratio of 1:1 in a 96‐well plate for 6 h. Luciferase activity was measured with a luminometer following the manufacturer's instructions (Promega, E2620).
For Fig 2H, JP‐luc cells were co‐cultured with Raji‐L1 or parental Raji cells in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 for 6 h and the supernatants were collected after 24 h. IL‐2 was quantified by ELISA using Human IL‐2 ELISA kit (Mlbio, HX2390).

The activities of superoxide dismutase, catalase, and glutathione peroxidase were respectively determined by a total SOD assay kit with WST-8, catalase assay kit, and cellular glutathione peroxidase assay kit with NADPH (Beyotime, Shanghai, China). Lipid oxidative damage marker malondialdehyde was detected using a lipid oxidation (MDA) assay kit (Beyotime, Shanghai, China). Protein carbonylation, DNA oxidative damage marker 8-OHdG, and IL-2 were detected using a protein carbonyl content detection kit (Solarbio, Beijing, China), human 8-OHdG ELISA kit, and human IL-2 ELISA kit (Mlbio, Shanghai, China). HaCaT cells were seeded into the 6-well cell culture plates at a density of 1 × 10⁵ cells/well. After treatment with different doses of BLF (10, 20, and 40 μg/mL) and 1 mM AAPH for 48 h, HaCaT cells were harvested and lysed; then, they were centrifuged at 12,000× g for 10 min at 4 °C. Take the supernatant for determination. All steps were performed according to the manufacturer’s instructions.