Journal: Frontiers in Physiology

Article Title: Facilitation of Reparative Dentin Using a Drug Repositioning Approach With 4-Phenylbutric Acid

doi: 10.3389/fphys.2022.885593

Figure Lengend Snippet: Immunohistochemistry staining with GRP78, HRD1 and MPO after 3 and 5 days pulp access preparation. Arrowhead indicates nucleic localization patterns of HRD1 immunostaining (E, L, S, Z). Boxes in A, C-D, F, H, J-K, M, O, Q-R, T, V, X-Y, A′ indicate enlarged view. Scale bars: A, C, F, H, J, M, O, Q, T, V, X, A’: 200 μm; B, D-E, G, I, K-L, N, P, R-S, U, W, Y-Z, B’: 50 μm.

Article Snippet: Primary antibodies were directed against NESTIN (1:400; cat. no. ab11306; Abcam), glucose regulatory protein 78 (GRP78; 1:400; cat. no. ab21685; Abcam), HRD1 (1:400; cat. no. NB100-2526; Novus Biologicals), MPO (1:200; cat. no. bs-4943R; Bioss), CD31 (1:100; cat. no. AF3628; R&D System), and TGF-β1 (1:100; cat. no. ab92486; Abcam), and the secondary antibodies used in the present study were biotinylated goat anti-rabbit or anti-mouse immunoglobulin G. Immunocomplexes were visualized using a diaminobenzidine tetrahydrochloride reagent kit (cat.no.

Techniques: Immunohistochemistry, Staining, Immunostaining

Journal: Veterinary Research

Article Title: Evaluation of the innate immune response of caprine neutrophils against Mycobacterium avium subspecies paratuberculosis in vitro

doi: 10.1186/s13567-023-01193-7

Figure Lengend Snippet: Gating strategy used for phagocytosis quantification. A Neutrophils, B Neutrophils with Map -GFP. In the first column, cells were gated based on FSC and SSC; in the second column, a doublet discrimination strategy was applied; in the third column, neutrophils were gated by myeloperoxidase positivity by fluorescence in the APC-Vio770 channel; in the fourth column, GFP + neutrophils were gated in the FITC channel and considered positive to phagocytosis of Map- GFP. Uninfected, non-incubated cells were used to ensure cell integrity and purity. Uninfected, incubated cells were used to adjust the neutrophil gate based on scatter parameters. This sample was also used to adjust the fluorescence thresholds of GFP and AF750. Flow cytometry data from 10 000 neutrophils from each animal ( n = 3) was analyzed using MACSQuantify™ software (Miltenyi Biotec Inc).

Article Snippet: After two washes with PBS, an overnight incubation with both the rabbit anti-MPO polyclonal Alexa Fluor 750 Conjugated (AF750) (BS-4943R-A750, Bioss, Woburn, MA, USA) [ ] at a 1:200 dilution, and the mouse anti-pan-histone primary antibody (MAB3422, Merck, Darmstadt, Germany) [ ] for NETs detection, at a 1:400 dilution was performed.

Techniques: Fluorescence, Incubation, Flow Cytometry, Software

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , Boxplots showing quantification of MPO–DNA and dsDNA (NET structures) in the plasma of HS ( n = 53) and patients with severe sepsis (with or without septic shock) ( n = 51). The boxes indicate the 25% quantile, median and 75% quantile. b , Scatterplots displaying correlations of platelet pyroptosis (activated caspase 1 positive platelets) with the levels of MPO–DNA (HS, n = 15; severe sepsis, n = 34) complexes and dsDNA (HS, n = 20; severe sepsis, n = 29) in the plasma of patients with severe sepsis (with or without septic shock) and HS ( r , correlation coefficient; n = 49). c , Immunofluorescence analysis showing S100A8/A9-induced pyroptotic platelets induced NET formation. Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (green) and CD41 for platelets (red). Scale bars, 25 μm and 7.5 μm. d , Quantification of MPO–DNA and dsDNA in the supernatant of cells ( n = 6). e , Immunofluorescence analysis showing NET formation in PMNs treated with rmS100A8/A9-induced platelets (WT or Tlr4 −/− ) (n = 6). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (red) and CD41 for platelets (green). Scale bars, 25 μm and 7.5 μm. f , Bar graphs displaying the levels of MPO–DNA and dsDNA in the supernatant of cells treated with rmS100A8/A9-induced platelets ( Tlr4 −/− or WT) ( n = 6). g , Levels of ox-mtDNA in supernatant of S100A8/A9-induced pyroptotic platelets (for 4 h) determined using a General 8-OHdG ELISA Kit ( n = 5). h , Levels of ox-mtDNA in plasma from sepsis patients (sepsis, n = 11; severe sepsis, n = 17) and HS ( n = 13). i , Levels of ox-mtDNA in plasma of sham (n = 7) or CLP mice ( n = 9). Data were presented as mean ± s.d. Statistical analysis was conducted using two-tailed Mann–Whitney test ( a ), two-tailed Pearson’s correlation test ( b ), one-way ANOVA and Tukey’s multiple comparisons test ( d , f , g ), Kruskal–Wallis test and Dunn’s multiple comparisons test ( h ) and two-tailed unpaired t -test ( i ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , S100A8/A9 released from NETs determined by ELISA ( n = 5). b , Representative immunofluorescence images of pyroptotic platelets incubated with PMA-treated murine PMNs (WT or S100a9 −/− ). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (cyan), CD41 for platelets (red) and caspase 1 for pyroptosis (green). Scale bars, 25 μm and 5 μm. c , Bar graphs displaying mitochondrial ROS production in platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) using FACS analysis ( n = 5). d , Immunofluorescence analysis showing coexpression of CD41 (green), ASC (red) and NLRP3 (blue) in platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) ( n = 5). Purple indicates overlap. Scale bars, 5 μm and 1 μm. e , Quantified results for the NLRP3 inflammasome are shown. f , Bar graphs displaying the caspase 1 activity of platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) by FACS analysis ( n = 5). Data were presented as mean ± s.d. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparisons test ( a , c , e , f ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Incubation, Staining, Activity Assay

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , PMNs ( S100a9 -/- or WT) were incubated with 50 nM PMA to induced NET formation for 4 hours, and then incubated with platelets for another 4 hours. Representative immunofluorescence of PMNs incubated with platelets. Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (cyan), CD41 for platelet (red) and activated caspase 1 for pyroptosis (green); scale bars: 25 μm and 5 μm. b , In the CLP-induced sepsis model, platelet counts in Gsdmd fl/fl PF4-Cre mice and littermate control Gsdmd fl/fl mice were assessed at 0, 2, 4, and 6 hours using a hematology analyzer (n = 5). Data was presented as mean ± SD. Two-way ANOVA and Tukey’s multiple comparisons test for b. Abbreviation is as follow: PLT, platelet; NS, not statistically significant; PMNs, polymorphonuclear neutrophils; PMA, phorbol myristate acetate; MPO, myeloperoxidase; Sham, sham-operated mice; CLP, CLP-induced sepsis mice; GSDMD, Gasdermin D.

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Incubation, Immunofluorescence, Staining

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , Immunofluorescence analysis showing NET formation induced by platelets from Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice treated with S100A8/A9 ( n = 5). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (red) and CD41 for platelets (green). Scale bars, 25 μm and 7.5 μm. b , Bar graphs displaying the levels of MPO–DNA and dsDNA in supernatant of rmS100A8/A9-induced platelets from Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice using PicoGreen fluorescent dye and MPO–DNA–ELISA, respectively ( n = 5). c – h , In the CLP-induced sepsis murine model, levels of NETs, heterodimer S100A8/A9 and proinflammatory cytokines in plasma of Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice were detected using ELISA kits after 6 h. c , d , Bar graphs displaying plasma levels of MPO–DNA complexes ( c ) and dsDNA ( d ) in mice ( n = 5). e , Bar graphs showing plasma levels of heterodimer S100A8/A9 in mice ( Gsdmd fl/fl + sham, n = 7; Gsdmd fl/fl + CLP, n = 8; Gsdmd fl/fl PF4-Cre+sham, n = 8; Gsdmd fl/fl PF4-Cre+CLP, n = 6). f – h , Bar graphs showing plasma levels of IL-1β ( f ), TNF-α ( g ) and IL-6 ( h ) in mice ( n = 5). i , Survival analysis of Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice in CLP-induced sepsis model for 7 d ( n = 11). Data were presented as mean ± s.d. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparisons test ( b – h ) and the log-rank (Mantel–Cox) test ( i ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay