Journal: Kidney Diseases

Article Title: Clinical and Experimental Insights into the Role of NETosis in IgA Nephropathy Pathogenesis

doi: 10.1159/000546343

Figure Lengend Snippet: Elevated serum NETosis markers in IgAN patients correlate with CitH3 and Gd-IgA1 levels, and increased NETosis markers in renal tissue. a–c Comparison of serum levels of Citrullinated Histone H3 (CitH3), myeloperoxidase (MPO), and neutrophil elastase (NE) between IgAN patients ( n = 16) and healthy controls (HC, n = 13). d–f Pearson correlation analysis between serum CitH3, MPO, NE, and Gd-IgA1 levels. g Immunofluorescence staining of CitH3 and MPO in kidney tissues from IgAN ( n = 5) and minimal change disease (MCD, n = 5) patients. h–i Quantitative analysis of immunofluorescence staining intensity in the kidneys. Unpaired t tests were used for group comparisons, and Pearson correlation was employed for correlation analysis. Data are presented as mean ± SEM.

Article Snippet: Bone marrow was harvested from healthy C57BL/6J mice, and neutrophils were isolated utilizing a mouse bone marrow neutrophil isolation kit (Solarbio, P8550).

Techniques: Comparison, Immunofluorescence, Staining

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: C5a promoted arterial thrombosis by triggering neutrophils to release NETs. A , B H&E staining of thrombus, cross-sections ( A ) and longitudinal sections ( B ). Scale bar = 100 µm. C Quantification of the thrombus size in cross-sections by H&E staining ( n = 5 each). D Thrombus weight ( n = 5 each). E The blood flow velocity in the LICA was dramatically decreased in the FeCl 3 -induced thrombus mice compared with the sham mice, and this change was reversed by PMX53. F Quantification of the blood flow velocity ( n = 4 each). G Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Thirty minutes before FeCl 3 exposure, PMX53 was administered by intraperitoneal injection. Scale bar = 75 µm. H Quantification of NET formation capacity shown as the ratio of NETs/neutrophil in LCCA cross-sections subjected to immunofluorescence staining (NaCl group = 3, PMX53 group = 4). I Representative images of immunofluorescence stainings of NET formation for DNA (SYTOX green), CitH3 (red), Ly6g(blue) in vitro after stimulation of C5a, and NET formation was attenuated by PMX53. Scale bar = 75 µm. J Quantification of NET formation capacity in vitro shown as the percentage of NET release, which was assessed by immunofluorescence staining ( n = 3 each). (K) NETosis was measured using a plate reader assay. NET release is expressed as percentage of total DNA ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Staining, Immunofluorescence, Injection, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Interactions among C5a, mitochondrial STAT3 and NETs. A , B Western blot analysis was performed to test the expression levels of mitochondrial STAT3 and p-STAT3 (Ser 727 ) in neutrophil-like cells cocultured with C5a, compared with the control. VDAC, a marker of mitochondria, was used as a loading control for mitochondria ( n = 5 each). C NET release in response to buffer or AG490 was measured using a plate reader assay ( n = 3 each). D Representative images of immunofluorescence staining for DNA (SYTOX green), CitH3 (red) and Ly6g (blue) in vitro after stimulation with AG490 showing the presence of NETs. Scale bar = 100 µm. E Quantification of NET formation capacity shown as the percentage of NET release in vitro after stimulation with buffer or AG490, as assessed by immunofluorescence staining. ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Marker, Immunofluorescence, Staining, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: The reduction in arterial thrombotic burden induced by PMX53 was abolished by AG490 in vivo. A , B H&E staining of thrombus, cross-sections A and longitudinal sections B . The thrombus area was reduced by PMX53, and AG490 abolished this effect. A Scale bar = 100 µm; B Scale bar = 200 µm. C Quantification of the thrombus size in cross-sections by H&E staining ( n = 5 each). D Thrombus weight ( n = 5 each). E Blood flow velocity in the LICA. AG490 reversed the increased in blood flow induced by PMX53. F Quantification of the blood flow velocity ( n = 4 each). G Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Scale bar = 75 µm. H Quantification of NET formation capacity shown as the ratio of NETs/neutrophil in LCCA cross-sections subjected to immunofluorescence staining (NaCl group = 3; PMX53 group = 4; PMX53 + AG490 group = 3; AG490 group = 4). Data are presented as mean ± SD

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: In Vivo, Staining, Immunofluorescence

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Visual summary: the effect of complement C5a in arterial thrombosis. In arterial thrombosis, C5a chemotactically attracts neutrophils to migrate towards the culprit site and triggers the release of NETs, which contribute to thrombosis by promoting coagulation and stabilizing clots. C5a-induced promotion of NET release is dependent on Mito-ROS production. C5a induces the Mito-ROS production by inhibiting mitochondrial STAT3 activity

Article Snippet: Mouse neutrophils were isolated from the bone marrow of tibias and femurs from healthy C57BL/6 J mice using the Neutrophil Isolation Kit (Solarbio, China) following the manufacturer’s instructions.

Techniques: Coagulation, Activity Assay

Journal: Gels

Article Title: Composite Hydrogel Modulates Intrinsic Immune-Cascade Neovascularization for Ocular Surface Reconstruction after Corneal Chemical Injury

doi: 10.3390/gels9090676

Figure Lengend Snippet: Materials to inhibit neutrophil extracellular traps (NETs) in vitro: ( a ) immunofluorescence of DNase I@CS-SilMA (SCD) co-cultured with NETs; ( b ) immunofluorescence of NET generation-rate statistics; ( c ) NET marker cf-DNA content in the culture medium; ( d – f ) Western blot (WB) analysis and semi-quantification of NET markers cit-H3 and MPO. ( n = 3, all data are expressed as mean ± standard deviation; *** p < 0.001; ns, not significant; groups were compared using one-way ANOVA and Tukey’s test).

Article Snippet: Neutrophils were extracted using the Animal Bone Marrow Neutrophil Extraction Kit (Solarbio, Beijing, China) and used immediately after resuspension in RPMI-1640 (Corning, New York, NY, USA) supplemented with 2% fetal bovine serum (FBS, Gibco, New York, NY, USA).

Techniques: In Vitro, Immunofluorescence, Cell Culture, Marker, Western Blot, Standard Deviation