Fig 1: a Immunofluorescence analysis showed approximately similar biomarker expression of hLMSCs in SM for ocular surface marker (Pax6+), stem-cell biomarkers (ABCG2+, P63α+, ABCB5+) and the mesenchymal biomarkers (VIM+, CD90+, CD105+, CD 34−, HLA-DR− and CD45−) with respect to the cells in control medium; b the expression of MSC markers in hLMSCs grown in both the media was quantified using flow cytometry. More than 97% of cells were positive for CD105, CD90, and CD73, whereas less than 1% showed expression for negative markers CD45 and HLA-DR and approx. 6% of total cells were positive for CD34; c graphical representation of flow cytometry data. Blue: DAPI; Scale: 50 µM; Magnification: × 20 (all other micrographs) and 20 µM (CD73 of DMEM/F12 with 2% FBS; × 40 magnification)
Fig 2: Neutrophils are activated (showing histone citrullination) in the circulation and myocardium of mice with collagen-induced arthritis (CIA).(A) Representative immunofluorescence images of isolated neutrophils from blood of mice with CIA or healthy control mice. Neutrophils from mice with CIA are pre-activated (H3Cit+; pink) and show a propensity for spontaneous NET formation. Representative microscopic picture of isolated neutrophils are shown (Scale bar 50 μm). Healthy control (left) and CIA (right). * = activated H3Cit+ neutrophil. (n = 6). (B) Graph illustrating, as a function of time, the neutrophil-to-lymphocyte ratio a marker of hyperinflammatory response, measured in peripheral blood in DBA/1 J mice with CIA and a healthy control, respectively (n = 6). (C) Gating and quantification of infiltrating Ly6G+/CD45+ and CD45+ cells using flow cytometry in healthy control and CIA myocardial tissue (n = 14). (D) Quantitative comparison of tissue levels of IL-1β an inflammatory cytokine-mediating fibrosis measured using ELISA (n = 9). (E) Representative LV sections of CIA mice and healthy control mice stained for DAPI+ (blue), Ly6G+ (red), and H3Cit+ (green) cells (scale bar: 100 μm) and a representative image of an activated neutrophil in the right panel with co-staining of Ly6G+ (red) and H3Cit+ (green) (scale bar 10 μm). Comparative analysis of the total number of Ly6G+ (red) and H3cit+ (green) cells in LV heart sections of CIA mice and healthy controls (n = 8). Data are mean ± SEM. (C, E) *P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U test (C, E) t test.
Fig 3: Inhibition of PAD4 dampens thromboinflammation by decreased neutrophil infiltration, neutrophil H3Cit expression and reduced endothelial activation.(A) Representative immunostainings of LV sections and quantification of double-positive (Ly6G+ [red] and H3cit+ [green]) cells indicative of activated neutrophils in the myocardium of mice with collagen-induced arthritis (CIA) treated with the vehicle and mice with CIA treated with the PAD4 inhibitor, respectively (scale bar: 50 μm) (n = 7–8). (B) Quantification of infiltrating CD45+ and LY6G+ cells in the myocardial tissue of mice with CIA treated with the vehicle and mice with CIA treated with the PAD4 inhibitor, respectively, assessed by flow cytometry (n = 8). (C) Representative LV sections stained with DAPI+ (blue) and VWF+ (green). Quantification of VWF deposition in the myocardium of mice with CIA treated with the vehicle and mice with CIA treated with the PAD4 inhibitor (scale bar: 200 μm). (D) Myocardial tissue levels of IL-1β in mice with CIA treated with the vehicle and mice with CIA treated with the PAD4 inhibitor. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired T test. (B) Wilcoxon matched pairs signed rank test.
Supplier Page from Abcam for Anti-CD45 antibody