Recombinant human g csf

E. coli (JM109 strain, B528410-0001; Sanger Biotech) was grown as previously described [38 (link)]. Lipopolysaccharide (LPS) was obtained from E. coli O111:B4 (L3024; Sigma). dHL-60 cells were infected with E. coli (1 × 10⁶ CFUs), stimulated with LPS (100 ng/ml), or treated with recombinant human G-CSF (100 ng/ml; R&D) for the indicated times.

Priming of neutrophils was performed as described elsewhere [37 (link)]. We firstly determined the optimal source and concentration of G-CSF by priming the cells with 5, 10, 50, 100 or 200 ng/mL of recombinant G-CSF from different sources: recombinant human G-CSF (Gibco, Burlington, ON, Canada), recombinant human G-CSF (R&D Systems, Toronto, ON, Canada) and recombinant porcine G-CSF (Raybiotech, Burlington, ON, Canada)(S5 Fig). We also included priming with recombinant porcine GM-CSF (R&D Systems, Toronto, ON, Canada). Regarding porcine GM-CSF, a concentration of 50 ng/mL induced neutrophil priming for IL-8 production (see results), suggesting that this concentration is sufficient to prime the cells. Therefore, for the experiments, seeded neutrophils were primed for 30 min at 37°C, 5% CO₂, with 50 ng/mL of recombinant porcine G-CSF (Raybiotech) or recombinant porcine GM-CSF (R&D Systems), or culture medium for the non-treated group. At the end of the incubation period, primed cells were immediately used for the experiments.