Journal: Nature Communications

Article Title: Neutrophils and galectin-3 defend mice from lethal bacterial infection and humans from acute respiratory failure

doi: 10.1038/s41467-024-48796-y

Figure Lengend Snippet: a Schematic of PA14 infection with or without LPS pre-treatment. Bacterial load showing reduced bacterial burden in the lung ( b ) and decreased peripheral dissemination ( c ) in LPS + PA14 mice compared to PA14 mice (4 h post-infection). n = 9 (PA14), 9 (LPS(1d)+PA14), and 10 (LPS(3d)+PA14) mice. Data were log transformed using log base 10 to adjust for differences in standard deviation prior to analysis. Data pooled from 3 independent experiments and analyzed using one-way ANOVA with the Dunn’s test. d Kaplan-Meier survival curve showing 100% mortality of PA14 infected mice within 18–20 h of infection, whereas 100% survival of LPS + PA14 group as monitored up to 14 days post infection. n = 8 (PA14 and LPS + PA14) mice per group. Data representative of 3 independent experiments and analyzed using Log-rank test. e Higher albumin levels in BALF of PA14 mice compared to LPS ± PA14 mice (mice pre-exposed to LPS for 3 days), BALF being collected 4 h post-infection from both groups of mice and also from naïve mice and mice only treated with LPS. n = 7 (Naïve and LPS), and 8 (PA14 and LPS + PA14) mice. Data pooled from 2 independent experiments and analyzed using ordinary one-way ANOVA with the Tukey post-hoc test. f RT-qPCR analysis of gene expression of Il1b , Il6 , Ifit1 , Isg15, Ifng, Il10 , Stat1 and Aoah in the lung tissue of the four groups of mice. n = 3 mice per group. Data representative of 3 independent experiments and analyzed using ordinary one-way ANOVA with the Dunnett’s test. g MPO and ELA2 protein levels associated with neutrophil influx and chemokine and cytokine levels in the BALF of the two groups of mice. n = 7 (Naïve and LPS), and 8 (PA14 and LPS + PA14) mice. Data pooled from 2 independent experiments and analyzed using ordinary one-way ANOVA with the Tukey post-hoc test. All data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data and exact P values are provided as a file.

Article Snippet: ELISA kits were used for mouse albumin (Abcam, ab108792), myeloperoxidase (MPO) (R&D Systems, DY3667), neutrophil elastase (ELA2) (R&D Systems, DY4517), CXCL2 (R&D Systems, DY452) and galectin-3 (R&D Systems, DY1197).

Techniques: Infection, Transformation Assay, Standard Deviation, Quantitative RT-PCR, Gene Expression

Journal: Pulmonary Circulation

Article Title: Didang decoction attenuates cancer‐associated thrombosis by inhibiting PAD4‐dependent NET formation in lung cancer

doi: 10.1002/pul2.12454

Figure Lengend Snippet: DD suppressed neutrophil counts through G‐CSF. (a, b) Comparison of red blood cell and hemoglobin levels in each group. (c, d) Peripheral neutrophil and monocyte counts were measured by an automated hematology analyzer. (e, f) Concentrations of G‐CSF and GM‐CSF in serum were assessed by ELISA. (g, h) Serum MPO and NE levels were detected by ELISA. ELISA, enzyme‐linked immunosorbent assay; G‐CSF, granulocyte colony‐stimulating factor; GM‐CSF, granulocyte‐macrophage colony‐stimulating factor; MPO, myeloperoxidase; NE, neutrophil elastase; ns, no significance. n = 5, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus Normal or Tumor group.

Article Snippet: The levels of MPO, NE, granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), TF, and thrombin‐antithrombin (TAT) complex in mouse serum, as well as the MPO level in neutrophil supernatants, were measured using various murine enzyme‐linked immunosorbent assay (ELISA) kits: the MPO ELISA kit (ab155458, Abcam), NE ELISA kit (E‐EL‐M3025, Elabscience), murine G‐CSF ELISA kit (EMCSF3X5, Invitrogen), murine GM‐CSF ELISA kit (BMS612, Invitrogen), murine TF ELISA kit (E‐EL‐M1163, Elabscience), murine TAT complex ELISA kit (ab108907, Abcam), and murine IL‐8 ELISA kit (ml063162, mlbio), following the standard protocols.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay

Journal: Pulmonary Circulation

Article Title: Didang decoction attenuates cancer‐associated thrombosis by inhibiting PAD4‐dependent NET formation in lung cancer

doi: 10.1002/pul2.12454

Figure Lengend Snippet: DD reduced the formation of NETs in vitro. The primarily isolated neutrophils were incubated with CM from LLC cells and/or treated with DD or DNase I. (a) MPO level in the neutrophil supernatants was measured by ELISA. (b–d) Immunofluorescence analysis and quantitative data for MPO, citH3, and NE levels in neutrophils. Nuclei were stained with DAPI. Scale bar: 100 μm. citH3, citrullinated histone H; CM, conditioned medium; DAPI, 4′,6‐diamidino‐2‐phenylindole; DD, Didang decoction; ELISA, enzyme‐linked immunosorbent assay; LLC, Lewis lung carcinoma; MPO, myeloperoxidase; NE, neutrophil elastase; NET, neutrophil extracellular trap. n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus RPMI‐1640 or CM group.

Article Snippet: The levels of MPO, NE, granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), TF, and thrombin‐antithrombin (TAT) complex in mouse serum, as well as the MPO level in neutrophil supernatants, were measured using various murine enzyme‐linked immunosorbent assay (ELISA) kits: the MPO ELISA kit (ab155458, Abcam), NE ELISA kit (E‐EL‐M3025, Elabscience), murine G‐CSF ELISA kit (EMCSF3X5, Invitrogen), murine GM‐CSF ELISA kit (BMS612, Invitrogen), murine TF ELISA kit (E‐EL‐M1163, Elabscience), murine TAT complex ELISA kit (ab108907, Abcam), and murine IL‐8 ELISA kit (ml063162, mlbio), following the standard protocols.

Techniques: In Vitro, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Allergy, Asthma & Immunology Research

Article Title: Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population

doi: 10.4168/aair.2020.12.1.42

Figure Lengend Snippet: Demographics of enrolled patients

Article Snippet: The sections were incubated with a 1:500 dilution of mouse anti-human neutrophil elastase primary antibody (R&D Systems, Minneapolis, MN, USA) for 60 minutes at room temperature.

Techniques:

Journal: Allergy, Asthma & Immunology Research

Article Title: Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population

doi: 10.4168/aair.2020.12.1.42

Figure Lengend Snippet: Analysis of risk factors for refractoriness

Article Snippet: The sections were incubated with a 1:500 dilution of mouse anti-human neutrophil elastase primary antibody (R&D Systems, Minneapolis, MN, USA) for 60 minutes at room temperature.

Techniques:

Journal: Allergy, Asthma & Immunology Research

Article Title: Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population

doi: 10.4168/aair.2020.12.1.42

Figure Lengend Snippet: Arrows are displayed for neutrophils-associated markers against the first 4 principal components. The dots are displayed for individual patients, and they are divided into three colors based on their disease control status; the refractory group consists of the partly controlled and uncontrolled patients. (A) Neutrophils-associated markers against the first 2 components. (B) Individual patients categorized by their disease control status against the first 2 components. (C) Neutrophils-associated markers against the third and fourth 2 components. (D) Individual patients categorized by their disease control status against the third and fourth 2 components. FAMD, factor analysis of mixed data; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; MPO, myeloperoxidase; MMP, matrix metallopeptidase; CXCL, chemokine (C-X-C motif) ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; OSM, oncostatin M; DCS, disease control status.

Article Snippet: The sections were incubated with a 1:500 dilution of mouse anti-human neutrophil elastase primary antibody (R&D Systems, Minneapolis, MN, USA) for 60 minutes at room temperature.

Techniques: Control

Journal: Allergy, Asthma & Immunology Research

Article Title: Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population

doi: 10.4168/aair.2020.12.1.42

Figure Lengend Snippet: NP, nasal polyp; HNE, human neutrophil elastase; MPO, myeloperoxidase; IL, interleukin; DAPI, 4′,6-Diamidino-2-phenylindole dihydrochloride.

Article Snippet: The sections were incubated with a 1:500 dilution of mouse anti-human neutrophil elastase primary antibody (R&D Systems, Minneapolis, MN, USA) for 60 minutes at room temperature.

Techniques:

Journal: Allergy, Asthma & Immunology Research

Article Title: Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population

doi: 10.4168/aair.2020.12.1.42

Figure Lengend Snippet: Imbalance of neutrophil elastase and α1 anti-trypsin in the refractory nasal polyps. (A) Concentration of α1 anti-trypsin between the controlled and the refractory groups. (B) The ratio of human elastase-positive cells/α1 anti-trypsin between the controlled and the refractory groups.

Article Snippet: The sections were incubated with a 1:500 dilution of mouse anti-human neutrophil elastase primary antibody (R&D Systems, Minneapolis, MN, USA) for 60 minutes at room temperature.

Techniques: Concentration Assay

Journal: Open Journal of Immunology

Article Title: The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

doi: 10.4236/oji.2022.124007

Figure Lengend Snippet: Figure 2. Serum levels of MPO, NE and DNA. (a) Serum levels of MPO in RA patients were signifi- cantly higher than those in HC (p < 0.0001). (b) Serum levels of NE in RA patients were significantly higher than those in HC (p < 0.0001). (c) Serum DNA levels in RA were significantly decreased compared to those of HC (p < 0.01). RA, rheumatoid arthritis; HC, healthy control; MPO, myelope- roxidase; NE, neutrophil elastase; DNA, deoxyribonucleic acid; RFU, relative fluorescence unit.

Article Snippet: We added 1:300 diluted rabbit anti-human neutrophil elastase antibody (diluted with 5% BSA solution), incubated overnight at 4 ̊C then 1:200 diluted Rhodamine (TRITC; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) labeled goat anti-rabbit IgG antibody for 1 h at room temperature in the dark.

Techniques: Control, Fluorescence

Journal: Open Journal of Immunology

Article Title: The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

doi: 10.4236/oji.2022.124007

Figure Lengend Snippet: Figure 3. Scatter plots demonstrating the correlations of SAA levels with MPO, NE and the association of MPO and NE with dis- ease activity in rheumatoid arthritis.: (a) SAA levels with MPO (R2 = 0.548; p < 0.0001); (b) SAA with NE (R2 = 0.131; p < 0.0001); (c) Serum MPO levels with CRP (R2 = 0.071; p = 0.0059); (d) MPO with ESR (R2 = 0.235; p < 0.001); (e) MPO with DAS28 score (R2 = 0.295; p < 0.0001); (f) MPO with anti-CCP (R2 = 0.005; p = 0.47); (g) MPO with RF IgG (R2 = 0.005; p = 0.45). (h) MPO with RF IgA (R2 = 0.007; p = 0.39); (i) Serum NE levels with CRP (R2 = 0.01; p = 0.18); (j) NE with ESR (R2 = 0.03; p = 0.06); (K) NE with DAS28 score (R2 = 0.093; p = 0.0015); (l) NE with anti-CCP (R2 = 0.006; p = 0.39); (m) NE with RF IgG (R2 = 0.002; p = 0.58). (n) NE with RF IgA (R2 = 0.0046; p = 0.48). SAA, serum amyloid A; MPO, myeloperoxidase; NE, neutrophil elastase; DAS28, dis- ease activity score for 28 joints; ESR, erythrocyte sedimentation rates; CRP, C reactive protein; anti-CCP, anti-cyclic citrullinated peptide; RF, rheumatoid factor; IgG, immunoglobulin G; IgA, immunoglobulin A; R2, correlation coefficient.

Article Snippet: We added 1:300 diluted rabbit anti-human neutrophil elastase antibody (diluted with 5% BSA solution), incubated overnight at 4 ̊C then 1:200 diluted Rhodamine (TRITC; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) labeled goat anti-rabbit IgG antibody for 1 h at room temperature in the dark.

Techniques: Activity Assay, Sedimentation

Journal: Open Journal of Immunology

Article Title: The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

doi: 10.4236/oji.2022.124007

Figure Lengend Snippet: Figure 5. Spontaneous NETs formation and enhanced NETs expression under stimulation in RA patients. (a) Neutrophils in RA patients were primed to form NETs with an enhanced expression after LPS or SAA stimulation. DAPI was used to stain neutro- phils’ nuclei in blue and the arrows indicated the web-like structures of the forming NETs. (b) Statistical measurements of NETs in each group. In a basal condition, NETs expression in RA group was statistically higher than that of HC group; Under LPS or SAA stimulation, the formed NETs were higher than that in HC. However, compared with LPS group, the NETs expression in- duced in SAA was significantly higher. The data were expressed as the percentage of NETs; *p < 0.05.

Article Snippet: We added 1:300 diluted rabbit anti-human neutrophil elastase antibody (diluted with 5% BSA solution), incubated overnight at 4 ̊C then 1:200 diluted Rhodamine (TRITC; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) labeled goat anti-rabbit IgG antibody for 1 h at room temperature in the dark.

Techniques: Expressing, Staining

Journal: Open Journal of Immunology

Article Title: The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

doi: 10.4236/oji.2022.124007

Figure Lengend Snippet: Figure 6. SAA induced NETs formation and increased DNA concentration was mediated by TLR4 activation. (a) immunofluo- rescence of NETs formation into the different groups. The overlap of NE staining (red) and DNA staining (blue) was used to represent the formed NETs. As indicated by the arrows, some neutrophils lost their original lobular nucleus or rod-shaped nuc- leus, and the DNA loosely diffused to form a fibrous network (blue), and there were NE staining positive parts (red), namely NETs (partial Purple), which were seen in the SAA and LPS groups; while the blue nuclei in the picture, surrounded by the red NE staining positive part, were normal neutrophils, more pronounced in (SAA+TAK-242) and (LPS+TAK-242). (b) quantitative measure of NETs formed into the different groups. Compared with the control group (5.734% ± 0.428%), the SAA stimulation group (14.58% ± 1.058%) significantly increased the formation of NETs (p < 0.05). Whereas, the pretreatment of neutrophils with TAK-242, and despite the same stimulation by SAA, the formation of NETs was significantly reduced (6.915% ± 0.418% vs. 14.58% ± 1.058%, p < 0.05). Similarly, the (LPS+TAK-242) group (6.905% ± 0.4646%) compared with the LPS group (13.71% ± 0.7951%), the formation of NETs was significantly reduced (p < 0.05). (c) DNA quantification in the neutrophils supernatants. There was a significantly lower DNA concentration in the culture supernatant of SAA group pretreated with TAK-242 (16.34 ± 0.611 μg/ml) compared with the SAA group (36.89 ± 1.288 μg/ml) (p < 0.05); As well, similar results were found in the LPS group (35.23 ± 0.7689 μg/ml) and (LPS+anti-TAK-242) group (15.77 ± 0.8893 μg/ml) (p < 0.05). There was no statistically significant change in the TAK-242 group and the control group (p > 0.05).

Article Snippet: We added 1:300 diluted rabbit anti-human neutrophil elastase antibody (diluted with 5% BSA solution), incubated overnight at 4 ̊C then 1:200 diluted Rhodamine (TRITC; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) labeled goat anti-rabbit IgG antibody for 1 h at room temperature in the dark.

Techniques: Concentration Assay, Activation Assay, Staining, Control

Journal: Open Journal of Immunology

Article Title: The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

doi: 10.4236/oji.2022.124007

Figure Lengend Snippet: Figure 7. Pathway analysis of NETs formation. (a) Morphologic observation of neutrophils exposed to stimulants in presence or not of NADPH inhibitors (apocynin or VAS2870). (b) Statistical analysis of NETs formation in the different groups. (c) Statistical analysis of DNA serum levels in the different groups. DAPI, 4', 6-diamidino-2-phenylindole; NE, neutrophil elastase; LPS, lipopo- lysaccharide; SAA, serum amyloid A. **** (p < 0.0001). < 0.05), but there was still a residual ability of endothelial cells to migrate (44.57% ± 1.798% vs. 16.57% ± 1.066%, p < 0.05) (Figure 8(b) and Figure 8(c)). What’s more, the tube formation assay was used to analyze the effect of NETs on the morphological angiogenesis process of HUVECs. The vascular tubule forma- tion in vitro was observed after 72 h of stimulation using an inverted micro- scope. The results showed that in the NETs (0.28 mg/l) stimulation group, there were obvious interconnections between cells to form tubules, and the tubules interconnected to form a complex network structure, suggesting that NETs can promote vascular tubules formation (2.857 ± 0.3401 vs. 1.143 ± 0.4041, p < 0.05). However, following NETs pretreatment (DNaseI), the tube formation was nearly unobserved (1.571 ± 0.3689 vs. 2.857 ± 0.3401, p < 0.05) (Figure 8(d) and Fig- ure 8(e)). Together, these results indicated that DNA components in NETs play an important role in the promotion of endothelial cells’ proliferation, migration, and vascular tube formation.

Article Snippet: We added 1:300 diluted rabbit anti-human neutrophil elastase antibody (diluted with 5% BSA solution), incubated overnight at 4 ̊C then 1:200 diluted Rhodamine (TRITC; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) labeled goat anti-rabbit IgG antibody for 1 h at room temperature in the dark.

Techniques: Tube Formation Assay, In Vitro, Migration

Journal: Open Journal of Immunology

Article Title: The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

doi: 10.4236/oji.2022.124007

Figure Lengend Snippet: Figure 8. NETs induced the proliferation, migration and the vascular tube formation of HUVECs. (a) NETs induced endothelial cells proliferation in vitro. Significant increases in proliferation of HUVECs stimulated with NETs (0.28 mg/l) were showed after 72 hours of incubation, while a significant decreases were observed after the addition of a mixture of extracted NETs and DNaseI (10 U/ml) for the same time of incubation. The data were expressed as the optical density (OD) measurement at 490 nm; *compared with control group, p < 0.05 versus #compared with NETs group, p < 0.05. (b) NETs induced endothelial cells migra- tion in vitro. The representative photomicrograph showed HUVECs repopulating the wound in response to NETs (0.28 mg/l). However, the pre-treatment with DNaseI (10 U/ml) displayed a significantly decreased migration of HUVECs. Original magnifi- cation: ×4. (c) Statistical results of the figure B; the data were expressed as the healing rate = (initial scratch area-current scratch area)/initial scratch area × 100%; *compared with control group, p < 0.05 versus #compared with NETs group, p < 0.05. (d) NETs induced HUVECs to form vascular tubes network. The representative photomicrograph showed that NETS (0.28 mg/l) induced HUVECs to form small complex interconnected tube vessels network over 72 hours of incubation, while they lack to interweave after their pre-treatment with DNaseI (10 U/ml). The arrows indicate the structure of blood vessels formed in vitro as the number of cross-nodes. Original magnification: ×10. (e) Statistical results of the figure D; the data were expressed as the number of cross nodes; *compared with control group, p < 0.05 versus #compared with NETs group, p < 0.05. joint disorder in RA patients. In the current work, we confirmed the high ex- pression of TLR4 in the synovial membranes, and elevated serum levels of SAA, in RA patients. Subsequently, our results indicated that SAA was associated to NETs components which correlated with disease activity. Moreover, neutrophils in RA were primed to undergo NETs formation. Latterly, we demonstrated NETs formation in ST of RA patients. Then, we indicated that SAA/TLR4 in- duced NETs formation and SAA-induced NETs formation was dependent on NADPH pathway. Finally, we demonstrated that extracted NETs could induce angiogenesis. We first showed that TLR4 was highly expressed in the synovial tissues of RA patients compared to OA patients. A previous study also reported that TLR4 was widely expressed in early and longstanding RA [38]. Moreover, as we previously demonstrated [39], this current research also showed that serum levels of SAA in

Article Snippet: We added 1:300 diluted rabbit anti-human neutrophil elastase antibody (diluted with 5% BSA solution), incubated overnight at 4 ̊C then 1:200 diluted Rhodamine (TRITC; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) labeled goat anti-rabbit IgG antibody for 1 h at room temperature in the dark.

Techniques: Migration, In Vitro, Incubation, Control, Activity Assay

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