Human monocyte nucleofection kit

pT7-EGFP-C1 (empty vector), pT7-EGFP-C1-HsDCP2 (wild-type), pT7-EGFP-C1-HsDCP2-S249D (phosphomimetic mutant), and pT7-EGFP-C1-HsDCP2-S249A (phosphodeficient mutant) were transformed using nucleofection into non-differentiated THP-1 monocytes (0.5 μg plasmid per 2.5×10⁶ cells) using Human Monocyte Nucleofection Kit (Lonza) as instructed in the “Efficient non-viral transfection of THP-1 cells” paper. 6 h following nucleofection, THP-1 cells were differentiated into macrophages via 50 nM PMA treatment for 3 h. Differentiated THP-1 cells were seeded into 12-well plates and treated with 20 ng/ml LPS overnight. The next morning, the supernatant fractions were harvested for IL1B ELISA quantification and the cells were lysed for LC3, GFP and actin immunoblotting. IL1B measurement was performed as instructed in the e-Bioscience human IL1B ELISA kit.

Murine neutrophils were purified from bone marrows as previously described (Zhang et al., 2010 (link)). Briefly, bone marrow cells collected from mice (8–10 weeks old) were treated with the ACK buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 127 µM EDTA) for red blood cell lysis, followed by a discontinuous Percoll density gradient centrifugation. Neutrophils were collected from the band located between 81% and 62% of Percoll. For transient transfection of primary mouse neutrophils, three million neutrophils were electroporated with 1.6 µg endotoxin-free plasmids or 300 nM of siRNA using the human monocyte nucleofection kit (Lonza, Switzerland) with an Amaxa electroporation system. The cells were then cultured for overnight in the medium supplied with the kit containing 10% FBS and 25 ng/ml recombinant GM-CSF (PeproTech, Rocky Hill, NJ). Cell sorting was done by a FACS Aria sorter (BD, San Jose, CA). We have used this method to overexpress protein in primary neutrophils for many years and electroporated neutrophils have good response to chemoattractant stimulation (Gao et al., 2015 (link); Tang et al., 2011 (link); Xu et al., 2010 (link); Zhang et al., 2013 (link)). And this method is also widely used by other groups (Loison et al., 2014 (link); Magalhaes et al., 2007 (link); Sun et al., 2007 (link)).

Murine neutrophils were purified from bone marrows as previously described (Zhang, et al., 2010 (link)). Briefly, bone marrow cells collected from mice were treated with the ACK buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 127 μM EDTA) for red blood cell lysis, followed by a discontinuous Percoll density gradient centrifugation. Neutrophils were collected from the band located between 81% and 62% of Percoll. Transient transfection of neutrophils were done as previously described (Yuan, et al., 2017 (link); Basit, et al., 2016 (link); Simunovic, et al., 2015 (link); Loison, et al., 2014 (link); Zhang, et al., 2013 (link); Tang, et al., 2011 (link); Xu, et al., 2010 (link); Zhang, et al., 2010 (link); Sun, et al., 2007 (link)). In brief, three million neutrophils were electroporated with 1.6 μg endotoxin-free plasmids or 300 nM of siRNA using the human monocyte nucleofection kit (Lonza, Switzerland) with an Amaxa electroporation system. The cells were then cultured for overnight in the medium supplied with the kit containing 10% FBS and 25 ng/ml recombinant GM-CSF (PeproTech, Rocky Hill, NJ). Cell sorting was done by a FACS Aria sorter (BD, San Jose, CA).