Neutrophil-like Differentiation and Transfection of HL-60 Cells

The cell line HL-60 (TCHU 23) was purchased from the Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were plated at 5 × 10⁵ per well cultured in IMDM with 20% FBS combined antibiotics (1:100) according to the recommendation on the official website of ATCC. HL-60 cells were treated with 1.25% DMSO for 4 days to acquire a neutrophil-like phenotype. The DMSO induced HL-60 was an approved model to study biological function of neutrophil as previous article showed59 (link)60 (link)61 (link). Then electrotransfections were exerted through 4D-Nucleofector™ System (Lonza Cologne GmbH 50829 Cologne, Germany) combined with Nucleofector™ Solution under the recommendations of SF Cell Line 4D-Nucleofector™ X Kit. The transfection efficiency was more than 50% which was confirmed through positive plasmid control 0.4 μg pmaxGFP™ Vector and the cell viability (% trypan blue negative cells) is usually around 60 % after 24 hours. The LC3-GFP plasmid was kindly provided by Dr Zhenhong Ni in our department which was generated as described before39 (link). The transfected cells were incubated in humidified 37 °C/5% CO₂ incubator for 24 hours and then were treated for determined conditions and observed under the fluorescence microscope (Olympus IX81, Tokyo, Japan) with 40 lens.