Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 1: Trigger-dependent Microvesicle Shedding. Scanning electron micrograph (a) and size-distribution assessed by NTA (b) of PMN-derived microvesicles originating from PMNs incubated with plasma-opsonized S. aureus bacteria, E. coli, LPS, heat-inactivated bacteria bioparticles or vehicle (HBSS). PMN-derived CD11β/CD18 and CD11β/CD177-double positive events assessed by flow cytometry as a function of bacterial triggering agent (n = 3) (c). Scanning (d,e) and transmission electron micrographs (f,g) of PMNs showing pronounced membrane budding and shedding of microvesicles following incubation with opsonised S. aureus particles for 30 minutes (arrow indicates S. aureus particle) (e,g) compared to PMNs incubated with HBSS (d,f). 3D-tomographies and outer surface reconstructions of PMN incubated with S. aureus further confirmed the constriction of vesicles from the outer membrane seen in TEM (h). Raman spectroscopy maps of PMN incubated with (top, I) or without (bottom, II, control) bacteria showed lipid droplets and peri-membranous accumulation of glycogen granules in stimulated PMNs (I) compared to control (II) (i). PMNs exposed to S. aureus compared to resting PMNs (Figure 1d,e). Transmission electron micrographs of thin sections of PMNs containing phagocytised S. aureus bacteria confirmed increased membrane budding and formation of microvesicles (Figure 1f,g). Formation of glycogen granule clusters, translocation and peri- membranous massing of glycogen granule aggregates, and shipping of cytoplasmatic microvesicles containing glycogen granules were observed in PMNs exposed to bacteria, while glycogen granules remained well-dispersed in the cytoplasm of unstimulated PMNs (Figure 1f,g). 3D-tomography of PMNs further confirmed

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Derivative Assay, Incubation, Clinical Proteomics, Bacteria, Flow Cytometry, Transmission Assay, Membrane, Raman Spectroscopy, Control, Translocation Assay, Tomography

Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 3. Microvesicles in Plasma Samples from an Experimental Sepsis Model. Caecal ligation and puncture (CLP) procedure in rats (a). Time-dependent concentration of neutrophil- derived CD11β/CD18-double positive microvesicles assessed by flow cytometry (b). Aggregation of S. aureus bacteria standard with microvesicle isolates from animal plasma at the 24 and 48 hour time point (c) and corresponding ROC curves (d). Characterization of Microvesicle-Bacteria Aggregates In order to better understand the nature of the microvesicle-bacteria aggregates, we used an in vitro analysis to further characterize their properties. The CD11β-positivity of the aggregating human PMN- derived vesicles was confirmed by immunostaining (Figure 4a) and transmission electron micrographs of microvesicle-bacteria aggregates were recorded (Figure 4b). The microvesicle- concentration dependence of bacteria aggregation was confirmed by serially diluting microvesicle isolates from PMNs exposed to S.

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Clinical Proteomics, Ligation, Concentration Assay, Derivative Assay, Flow Cytometry, Bacteria, In Vitro, Immunostaining, Transmission Assay

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Immunofluorescence and western blotting results showing the expression of relevant cytokines in the CIBT of 2VO + EMS rats. A Representative triple immunofluorescence staining images showing ANGPT2 + , Tie2 + , and CD11b + cells in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. B Counts of ANGPT2 + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). C Counts of TEMs (Tie2 + and CD11b + ) in each group (n = 9, one-way ANOVA and Šídák's multiple comparisons test). D Representative western blot showing the relative expression of TRPS1, ANGPT2, Tie2, CD11b, VEGFA, IGF1, and CD31 in the CIBT adjacent to the TM tissue in each group (normalized to β-actin expression). E Densitometric analyses of the relative expression of TRPS1, ANGPT2, Tie2, CD11b, VEGFA, IGF1, and CD31 (n = 3, one-way ANOVA and Šídák's multiple comparisons test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). The error bars represent the ± SDs. CIBT: chronically ischaemic brain tissue; EMS: encephalomyosynangiosis; TEMs: Tie2-expressing monocytes/macrophages; 2VO: 2-vessel occlusion

Article Snippet: After being washed with PBST, the slices were incubated with 488-conjugated goat anti-rabbit (1:2000, ab150077, Abcam, UK), 594-conjugated goat anti-mouse (1:2000, ab150116, Abcam, UK), and 647-conjugated donkey anti-goat (1:2000, ab150135, Abcam, UK) secondary antibodies at room temperature in a dark room for 2 h. The 647-conjugated CD11b antibody (0.5 μg, CL647-65229, proteintech, China) was used to directly label CD11b.

Techniques: Immunofluorescence, Western Blot, Expressing, Staining

Journal: Cell & Bioscience

Article Title: Tie2-expressing monocytes/macrophages promote angiogenesis in chronically ischaemic brain tissue

doi: 10.1186/s13578-025-01401-1

Figure Lengend Snippet: Immunofluorescence results showing the expression of pro-angiogenesis factors in the CIBT of 2VO + EMS rats. A Representative triple immunofluorescence staining images showing Tie2 + , CD11b + , and VEGFA + cells and in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. B Counts of VEGF + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). C Representative triple immunofluorescence staining images showing Tie2 + , CD11b + , and IGF1 + cells in the CIBT in each group (white arrows indicate TEMs). Bar = 50 µm. D Counts of IGF1 + cells in each group (n = 3, one-way ANOVA and Šídák's multiple comparisons test). CIBT: chronically ischaemic brain tissue; EMS: encephalomyosynangiosis; TEMs: Tie2-expressing monocytes/macrophages; 2VO: 2-vessel occlusion

Article Snippet: After being washed with PBST, the slices were incubated with 488-conjugated goat anti-rabbit (1:2000, ab150077, Abcam, UK), 594-conjugated goat anti-mouse (1:2000, ab150116, Abcam, UK), and 647-conjugated donkey anti-goat (1:2000, ab150135, Abcam, UK) secondary antibodies at room temperature in a dark room for 2 h. The 647-conjugated CD11b antibody (0.5 μg, CL647-65229, proteintech, China) was used to directly label CD11b.

Techniques: Immunofluorescence, Expressing, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Neutrophil membrane-coated circular RNA nanoparticles for targeted immunotherapy in HER2-positive breast cancer brain metastasis

doi: 10.1186/s12964-025-02321-w

Figure Lengend Snippet: Construction and Characterization of HER2-Targeted dHL-60 Cell Membranes. (A) Schematic representation of the lentiviral vector design used to overexpress the scFv targeting HER2. The vector includes the anti-HER2 scFv, a membrane localization signal, a FLAG tag for detection, a hinge region for flexibility, and a transmembrane domain to anchor the scFv to the cell membrane. (B) SPR analysis illustrating the binding affinity of scFv to the HER2 protein. (C) Flow cytometry analysis comparing the binding of scFv (anti-HER2) to SKBR3 cells versus a control scFv. (D) Flow cytometry assessment of CD11b expression levels in HL-60 cells before and after differentiation induction with DMSO. (E) Western blot results demonstrating the expression of adhesion molecules CD11b, CD18, CD49d, and CD31 in differentiated HL-60 cells. (F) Western blot analysis was performed using an anti-FLAG antibody to detect the FLAG-tagged scFv (anti-HER2). A band corresponding to the scFv-FLAG fusion protein is observed in dHL-60 cells transduced with the lentiviral vector, but not in control cells. (G) Flow cytometry analysis (anti-FLAG antibody) demonstrating the surface expression of scFv (anti-HER2) on dHL-60 cells overexpressing scFv (anti-HER2). (H) Illustration depicting the construction and extraction process of the cell membrane from HL-60 cells, outlining the steps involved in preparing membrane-coated nanoparticles

Article Snippet: CoraLite ® Plus 488, PE-conjugated Goat Anti-Rabbit IgG, antibodies against CD11B, CD49d, CD31, CD18, β-actin, His Tag, FLAG Tag and CXCL9 were obtained from Proteintech (Wuhan, China).

Techniques: Plasmid Preparation, Membrane, FLAG-tag, Binding Assay, Flow Cytometry, Control, Expressing, Western Blot, Transduction, Extraction