IL-15/IL-15Rα protein was transiently transfected and expressed by using FreeStyle 293 cells (GIBCO, Cat#R79007). FreeStyle 293 cells were cultured in Freestyle 293 expression medium (GIBCO, Cat#12338018), supplemented with Ultra Low IgG Fetal Bovine Serum (ultra low immunoglobulins FBS, GIBCO, Cat # 16250078) at a final concentration of 1%. IL-15/IL-15Rα expression plasmids and transfection reagent PEI (Polysciences, Cat#239662) were prepared, the two plasmids of IL-15 and IL-15Rα were co-transfected at a ratio ranging from 1:1 to 9:1, total amount of plasmids was 100 μg/100 ml cells, the ratio of plasmid to PEI was 1:2 by mass. Cell density on the day of transfection was 1 × 106/ml. 1 L of FreeStyle 293 cells were prepared to be transfected. 50 ml of Opti-MEM (GIBCO, Cat # 11058021) medium was mixed with the plasmid, kept still for 5 min and filtered. Another 50 ml of Opti-MEM medium was mixed with PEI, kept still for 5 min and filtered. The plasmid was mixed with PEI and kept still for 15 min. The mixture of plasmid and PEI was slowly added to the cells and cultured in shaking incubator at 130 rpm at 37 °C, 8% CO2. 5 days later, the supernatant was collected by centrifugation for protein purification.
Ref1 molecule was prepared as previously reported23 (link).
Ref1 molecule was prepared as previously reported23 (link).