Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: The percentage of the neutrophil extracellular trap (NET) area in the SS and FS plots of flow cytometry was compared among untreated neutrophils and samples following NET induction with E. coli DH5α or PMA. (a) Expression intensities of Cit-H3, flavocytochrome b558, SYTOX™ Green, CD14, TLR2, TLR4, and LL-37 were compared between untreated neutrophil and NET areas after E. coli DH5α stimulation. (b) NET ratios were compared among untreated neutrophils and samples treated with E. coli DH5α or PMA using enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. ** p < 0.01. (c) Western blotting was performed to examine CD14 and Cit-H3 protein levels in untreated neutrophils and those stimulated with E. coli DH5α or PMA. Band intensities were quantified using ImageJ and normalized to β-actin. Data are presented as mean ± standard deviation (SD) (n = 3). Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s HSD test. ** p < 0.01

Article Snippet: Details of the antibodies used in this study and their sources are as follows: FITC-conjugated anti-human PR3 mouse monoclonal antibody (Cat. No. ab65255; Abcam, Cambridge, UK), PE-conjugated anti-human cathepsin D mouse monoclonal antibody (Cat. No. sc-13148 PE; Santa Cruz Biotechnology, Dallas, TX, USA), FITC-conjugated anti-human neutrophil elastase mouse monoclonal antibody (Cat. No. sc-55549 FITC; Santa Cruz Biotechnology, Dallas, TX, USA), PE-conjugated anti-human flavocytochrome b558 mouse monoclonal antibody (Cat. No. D162-5; Medical & Biological Laboratories, Tokyo, Japan), PE/Cyanine7-conjugated anti-human CD14 mouse monoclonal antibody (Cat. No. 367111; BioLegend, San Diego, CA, USA), FITC-conjugated anti-human CD68 mouse monoclonal antibody (Cat. No. F7135; DAKO, Glostrup, Denmark), FITC-conjugated anti-human CD282 (TLR2) mouse monoclonal antibody (Cat. No. 309705; BioLegend, San Diego, CA, USA), PE-conjugated anti-human CD284 (TLR4) mouse monoclonal antibody (Cat. No. 312805; BioLegend, San Diego, CA, USA), anti-citrullinated histone H3 (Cit-H3) (citrulline R2 + R8 + R17) rabbit polyclonal antibody (Cat. No. ab5103; Abcam, Cambridge, UK), anti-human LL-37 mouse monoclonal antibody (Cat. No. sc-166770; Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin rabbit monoclonal antibody (Cat. No. 4970; Cell Signaling Technology, Danvers, MA, USA), anti-human CD14 mouse monoclonal antibody (Cat. No. 14-0149-82; Invitrogen, Waltham, MA, USA), anti-PR3 mouse monoclonal antibody (Cat. No. sc-74534; Santa Cruz Biotechnology, Dallas, TX, USA), anti-human presepsin mouse monoclonal antibody (F1106-13–3; Mochida Pharmaceutical, Tokyo, Japan), anti-human presepsin rabbit monoclonal antibody (S68; Mochida Pharmaceutical, Tokyo, Japan), tetramethylrhodamine (TRITC)-conjugated goat anti-mouse IgG (H+L) secondary antibody (Cat. No. SA00007-1; Cosmo Bio, Tokyo, Japan), TRITC-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. SA00007-2, Cosmo Bio, Tokyo, Japan), Alexa Flour 405-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. ab175652; Invitrogen, Waltham, MA, USA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) secondary antibody (Cat. No. 7076; Cell Signaling Technology, Danvers, MA, USA), HRP-conjugated gort anti-rabbit IgG (H+L) secondary antibody (Cat. No. 7074; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot