Journal: bioRxiv

Article Title: Dynamic interaction between stress hormones and neutrophils promotes neutrophil extracellular trap formation with behavioral consequences

doi: 10.1101/2025.11.12.688017

Figure Lengend Snippet: Chronic restraint stress promotes NET formation. (A) Schematic illustration of the chronic restraint stress (CRS) model in mice. (B) Plasma corticosterone levels measured at baseline (day 0), day 14, and day 21 in control and CRS mice. (C) Plasma NET levels quantified as extracellular DNA or elastase levels (D) at day 28. (E) Splenic neutrophil infiltration at day 28. (F-G) NET-forming capacity of blood neutrophils isolated from control and CRS mice under basal and PMA-stimulated conditions. White arrows depict NETs. Data represent mean ± SEM (n = 5 mice/group). *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: NETs released in culture were quantified by measuring the fluorescence intensity of extracellular DNA using 1 μM Sytox Green (S7020, Invitrogen, Waltham, MA, USA) for 5 min. For microscopy studies, cultured cells were fixed, stained with sytox green or neutrophil elastase antibody (sc55549, Santa Cruz Biotechnology, Dallas, TX, USA) and imaged using a Zeiss LSM 710 confocal or Zeiss widefield microscope (Carl Zeiss Microscopy, Jena, Germany).

Techniques: Clinical Proteomics, Control, Isolation

Journal: bioRxiv

Article Title: Dynamic interaction between stress hormones and neutrophils promotes neutrophil extracellular trap formation with behavioral consequences

doi: 10.1101/2025.11.12.688017

Figure Lengend Snippet: Stress hormones induce NET formation. Neutrophils purified from human peripheral blood were cultured with varying concentrations of cortisol (A-C) or epinephrine (D-F) with or without PMA activation. NETs released in culture were quantified by sytox green fluorescence. NETs were visualized by confocal microscopy as the colocalization of extracellular DNA and neutrophil elastase. White arrows depict NETs. ***p < 0.001, ****p < 0.0001.

Article Snippet: NETs released in culture were quantified by measuring the fluorescence intensity of extracellular DNA using 1 μM Sytox Green (S7020, Invitrogen, Waltham, MA, USA) for 5 min. For microscopy studies, cultured cells were fixed, stained with sytox green or neutrophil elastase antibody (sc55549, Santa Cruz Biotechnology, Dallas, TX, USA) and imaged using a Zeiss LSM 710 confocal or Zeiss widefield microscope (Carl Zeiss Microscopy, Jena, Germany).

Techniques: Purification, Cell Culture, Activation Assay, Fluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: Dynamic interaction between stress hormones and neutrophils promotes neutrophil extracellular trap formation with behavioral consequences

doi: 10.1101/2025.11.12.688017

Figure Lengend Snippet: NET-forming neutrophils release stress hormones and upregulate glucocorticoid receptor (GR) and β2-adrenergic receptor (β2AR) expression. (A-B) ELISA quantification of cortisol levels (A) and epinephrine levels (B) in culture supernatants of neutrophils stimulated with PMA versus untreated controls. (C–D) Flow cytometry analysis of surface GR expression at 4 h post-PMA stimulation. (E–F) Flow cytometry analysis of β2AR expression at 4 h post-PMA stimulation. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: NETs released in culture were quantified by measuring the fluorescence intensity of extracellular DNA using 1 μM Sytox Green (S7020, Invitrogen, Waltham, MA, USA) for 5 min. For microscopy studies, cultured cells were fixed, stained with sytox green or neutrophil elastase antibody (sc55549, Santa Cruz Biotechnology, Dallas, TX, USA) and imaged using a Zeiss LSM 710 confocal or Zeiss widefield microscope (Carl Zeiss Microscopy, Jena, Germany).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry