The Netosis Formation of HL-60 Cell Differentiated to Neutrophil-Like Cells by LPS

Abstract

Journal homepage: www.zums.ac.ir/jhehp Background: Neutrophils are the most abundant white blood cells in humans. Recently, a novel strategy called the formation neutrophil extracellular traps (NETs) was described. NETs is a new strategy for pathogen response. This study focused on whether LPS induced NETs release in vitro in the HL60 cell line. Methods: In this study, the HL60 cell line was used for culture and DMSO for induction and differentiation. Flow cytometry was used to evaluate CD11b in the differentiated cells, and the NBT assay was used to evaluate the functionality of the differentiated HL-60 cells. Neutrophil-like cells were incubated with LPS (200 ng/ml) for 45 min, followed by incubation for 25 min with 100 ng/ml Hoechst 33342. Trypan blue as vital staining was used for viability. The statistical significance of the difference between the control and treated groups was evaluated using a one-way ANOVA. Results: Our results showed that 75% NETs was produced by HL-60 differentiated neutrophil cells exposed to 200 ng/ml LPS in 45 minues. Conclusion: Consequently, the LPS-induced infection and lethality may occur through various mechanisms. Thus, understanding the molecular mechanisms regulating NET formation in LPS-induced neutrophil-like cells would support the development of new therapeutic methods.

Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19

“…Notably, dHL-60 cells have previously been used to study NETosis in vitro. 31,56 Consistent with those prior reports, R848 induced NETosis in dHL-60 cells (Figure S7). Furthermore, we observed that pS9L inhibited NETosis and that siRNA knockdown of Siglec-9 (encoded by SIGLEC9) or SHP-1 (encoded by PTPN6) abrogated the effect of pS9L (Figures 3e, S8 and S9).…”

Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19

“…The HL-60 line is a promyelocytic leukemia that can be differentiated into a neutrophil-like cells (dHL-60) using all-trans retinoic acid (ATRA, 100 nM) and dimethylsulfoxide (DMSO, 1.25% v/v), which have previously been used to study NETosis in vitro. 31,56 We observed that R848 induced NETosis in dHL-60 cell's ( Figure S7). Furthermore, we observed that pS9L inhibited NETosis and that siRNA knockdown of Siglec-9 (encoded by SIGLEC9) or SHP-1 (encoded by PTPN6) abrogated the effect of pS9L (Figure 3e, S8, S9).…”

Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19

“…The HL-60 line is a promyelocytic leukemia that can be differentiated into a neutrophil-like cells (dHL-60) using all-trans retinoic acid (ATRA, 100 nM) and dimethylsulfoxide (DMSO, 1.25% v/v), which have previously been used to study NETosis in vitro. 31,56 We observed that R848 induced NETosis in dHL-60 cell's (Figure S7). Furthermore, we observed that pS9L inhibited NETosis and that siRNA knockdown Sera and plasma from COVID-19 patients have been shown to be sufficient to induce NETosis of neutrophils isolated from healthy donors in vitro.…”