pIL-15

Protocol tips

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	For transfection, cells reached exponential stage were spun down at 1000 rpm, 5 min and re-suspended at a density of 3 × 106 cells/mL. Then the IL-15-expression plasmid in combination with sIL-15Rα/Fc-expression plasmids were mixed at 1:1 and co-transfected using linear polyethylenimine (PEI) with a molecular weight of 25 kDa (Polysciences; Warrington, PA, USA) as we described previously [22].

Protocol tips
For transfection, cells reached exponential stage were spun down at 1000 rpm, 5 min and re-suspended at a density of 3 × 106 cells/mL. Then the IL-15-expression plasmid in combination with sIL-15Rα/Fc-expression plasmids were mixed at 1:1 and co-transfected using linear polyethylenimine (PEI) with a molecular weight of 25 kDa (Polysciences; Warrington, PA, USA) as we described previously [22].

Publication protocol

The suspension HEK293E cells were cultured in a 1:1 mixture medium of Freestyle293 (Gibco; Waltham, MA, USA) and SFM4HEK293 (HyClone; Logan, Utah, USA) supplemented with 2% FBS and 100 μg/mL of G418 (Gibco) in shake flasks (Corning; Corning, NY, USA) at 37 °C with 125 rpm rotation and 5% CO2. Cell growth and viability were monitored using a cell counter (Countstar; Shanghai, China). For transfection, cells reached exponential stage were spun down at 1000 rpm, 5 min and re-suspended at a density of 3 × 106 cells/mL. Then the IL-15-expression plasmid in combination with sIL-15Rα/Fc-expression plasmids were mixed at 1:1 and co-transfected using linear polyethylenimine (PEI) with a molecular weight of 25 kDa (Polysciences; Warrington, PA, USA) as we described previously [22]. Supernatants were harvested after 6 days to collect the secreted IL-15·sIL-15Rα/Fc superagonist. At the same time, sIL-15Rα/Fc was produced as a control by transfecting HEK293 cells with sIL-15Rα/Fc-expression plasmid alone.

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