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

Journal: bioRxiv

Article Title: Elastase mediated white matter damage in cerebral small vessel disease: Microglia - neutrophils pas de deux

doi: 10.1101/2024.12.23.630204

Figure Lengend Snippet: (A) ELANE concentration in the mouse corpus callosum at different time points after BCAS surgery evaluated with ELISA. n = 8–9 mice per group. (B) ELANE catalytic activity in mouse corpus callosum homogenate samples at different time points after BCAS surgery measured by spectrophotometry. n = 8–9 mice per group. (C) ELANE expression in microglia was assessed by flow cytometric analysis at 7 days after BCAS or sham surgery. Quantifications of flow cytometry are displayed in the adjacent graph. n = 6–9 mice per group. (D) ELANE expression in the bone marrow, and MBP expression in the corpus callosum determined by western blot at 3 days, 7 days, 14 days, and 28 days after BCAS or sham surgery. β-actin served as an internal loading control. (E) Quantification of the intensity of ELANE expression in the bone marrow performed with ImageJ. n = 3 replicates per group. (F) Plasma ELANE concentration at different time points after BCAS or sham surgery evaluated with ELISA. n = 8–9 mice per group. See also Figure S1.

Article Snippet: ELANE expression in mouse corpus callosum areas and plasma samples was measured using commercial ELISA kits (MELA20, R&D Systems).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Spectrophotometry, Expressing, Flow Cytometry, Western Blot, Control, Clinical Proteomics

Journal: Cell Reports Medicine

Article Title: Modeling antithymocyte globulin-induced microvasculopathy using human iPSC-derived vascularized liver organoids

doi: 10.1016/j.xcrm.2025.102433

Figure Lengend Snippet: ATG induces delayed proinflammatory phenotypes of iLSECs with neutrophil recruitment and luminal degranulation (A) Volcano plot showing differentially expressed genes between ATG- or rIgG-treated iLSECs (24 h). (B) Gene set enrichment analysis (GSEA) of the “overview of proinflammatory and profibrotic mediators” pathway in ATG versus rIgG at 24 h. (C) ELISA quantification of CXCL1 levels in iLSEC supernatants after 24-h treatment with increasing concentrations of rIgG or ATG (5–500 μg/mL) (mean ± SD, n = 4 per group; Sidak’s multiple comparisons test). (D) Intravital confocal images of GFP + iLSEC-lined vessels (green) in the transplanted HLBO 6 and 24 h post-injection of rIgG or ATG. Blood flow and neutrophils are visualized with TRITC 2,000 kDa dextran (blue) and anti-Ly6G antibody (red). Scale bars, 20 μm. (E) Counts of Ly6G + neutrophils in the view fields at −1, 6, and 24 h post-injection of rIgG or ATG (mean ± SD, n = 6 fields from 2 mice per group; Sidak’s multiple comparisons test). (F) Intravital confocal images of GFP + iLSEC-lined vessels (gray) 6 h post-injection of rIgG or ATG, showing blood flow (TRITC 2,000 kDa dextran, blue), neutrophils (anti-Ly6G antibody, green), and neutrophil elastase (NE) (anti-NE antibody, red). Scale bars, 50 μm (merged panels, left) and 20 μm (cropped panels, right). White arrows indicate peri-neutrophil NE. (G) Quantification of NE-positive area normalized to iLSEC vascular area (mean ± SD, n = 3 fields from 1 mouse for rIgG, n = 6 fields from 2 mice for ATG; unpaired t test). (H) Immunohistochemical staining of NE in the liver from the ATG-associated SOS patient at day −13 (before ATG treatment), day 12 (during liver injury), and day 100 (after recovery). Images from zone 3 are shown. CVs are indicated. Scale bars, 20 μm. (I) Quantification of NE + neutrophils in liver zone 3 ( n = 9–13 per group; Tukey’s multiple comparisons test). See also , , , and .

Article Snippet: Neutrophil Elastase (E6K6Q) Rabbit mAb (Alexa Fluor 647 Conjugate) , Cell Signaling Technology , Cat# 79565; RRID:N/A.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Immunohistochemical staining, Staining