Gene pulser mxcell electroporation system

K562 cells (1 × 10⁶/ml) were electroporated with 1 μg of scrambled oligonucleotides or miRNA mimics (Ambion/Thermo Fisher Scientific, Austin, TX, USA) using the Gene Pulser MXcell electroporation system (Bio-Rad Laboratories, Hercules, CA, USA) using the condition described previously [18 (link)] and then cultured at 37 °C with a humidified atmosphere containing 5% CO₂ for 24 h or 48 h for further analysis of miRNA expression or for Western blot analysis, respectively.

U937 cells were purchased from the European Collection of Cell Cultures (Salisbury, UK) and electroporated with control CRISPR–Cas9 plasmids or CRISPR–Cas9 plasmids containing gRNA that targeted PADI2 (Santa Cruz biotechnology, Dallas, TX, USA) using the Gene Pulser MXcell electroporation system (Bio-Rad Laboratories, Hercules, CA, USA) under the condition of voltage 210 V and capacitance 960 μF. The cells were then cultured in Iscove’s Modified Dulbecco’s medium (IMDM) (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) with 0.3 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA). After drug selection, the surviving cells were isolated and validated by Western blotting. The U937 cells was then introduced to differentiate with “differentiated macrophages” by coculturation with 500 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere containing 5% CO₂ for 48 h. The differentiated macrophages were cocultured with LPS (20 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. The culture supernatants were then collected and stored at −80 °C for ELISA.