How to prepare IL-2/JES6-1 and IL-2/S4B6 complexes for in vivo studies

Required reagents:

rmIL-2 (for example from Peprotech or Miltenyi Biotech)

anti-IL-2 mAb clone JES6-1A12 (for example from BioXcell) for in vivo use, i.e. sterile without azide or any other preservative

anti-IL-2 mAb clone S4B6 (for example from BioXcell) for in vivo use, i.e. sterile without azide or any other preservative

sterile PBS

Prepare an IL-2 stock solution from lyophilized rmIL-2 (100 μg/ml) according to the manufacturer´s recommendations. Calculate the total amount of IL-2 complexes needed for Your experiments (in terms of IL-2 equivalent). Calculate the total amount of anti-IL-2 mAb required for the given amount of IL-2; the molar ratio must be 2:1 (IL-2:anti-IL-2 mAb). Add calculated amount of undiluted rmIL-2 (stock solution) into sterile 5 ml eppendorf tube. Add calculated amount of undiluted anti-IL-2 mAb into the tube and gently mix. Let form the complexes at room temperature for 15 min. Dilute the IL-2 complexes with sterile PBS to desired concentration. Typically, we inject 1.5 – 2.0 μg of IL-2 equivalent per one dose in 250 μL. Aliquot the IL-2 complexes and either directly use them or store them at -20 ºC for up to 6 months. You can freeze/thaw the IL-2 complexes once, but not repeatedly!