Journal: The Journal of Experimental Medicine

Article Title: A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis

doi: 10.1084/jem.20070432

Figure Lengend Snippet: Structural requirements for CRIg-Fc inhibition of the AP C3 and C5 convertases in mouse serum. (A) Left: Overlay of human (white) and murine (yellow) CRIg IgV domains. Side chains of residues M86, D87 (red) and H57, and Q59 (blue) are indicated. Middle: Surface representation of murine CRIg crystal structure. Indicated in green are surface-exposed amino acid residues that are not conserved in human and murine CRIg. Right: Surface representation of muCRIg. Indicated are amino acid residues that are substituted by alanine. Red, M86 and D87 (CRIg(A)); blue, H57 and Q59 (CRIg(B)). (B) CRIg-Fc, but not CRIg(A)-Fc, CRIg(B)-Fc, or control Fc-protein, inhibits the generation of C3a des Arg (left) and C5a des Arg (right) in zymosan-activated serum. Zymosan particles were incubated for 45 min at 37°C with 4% mouse serum in the presence of Mg 2+ and EGTA. C3a des Arg and C5a des Arg were detected by ELISA and expressed as percentages of values in the absence of recombinant protein. Results are representative of four independent repeats. (C) Murine CRIg-Fc inhibits complement activation through the alternative, but not classical, pathways. Rabbit erythrocytes (left) or IgM-oponized sheep erythrocytes (right) were exposed to C1q-deficient (left) or factor B–depleted (right) human serum in the presence of increasing concentrations of CRIg-Fc or control-Fc protein. Values expressed as percent hemolysis in the absence of inhibitors (mean ± SEM; n = 3).

Article Snippet: C3a des Arg levels were quantified with capture and detection antibodies from BD Biosciences ( ) or with a mouse C3a des Arg ELISA kit (Bachem; ).

Techniques: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis

doi: 10.1084/jem.20070432

Figure Lengend Snippet: CRIg-Fc inhibits AP complement activation induced by anti-collagen antibodies in vitro and in vivo. (A) CRIg-Fc inhibits the alternative pathway of complement induced by immune complexes in mouse serum. Collagen-coated microtitre plates were incubated with anti-collagen antibodies, and the wells were incubated with 10% mouse serum. Binding of C3 fragments to the microtitre plates was determined by ELISA. The experiment was repeated three times with similar results. (B) CRIg-Fc, but not CRIg(A)-Fc and control-Fc protein, inhibits C3a and C5a production in mouse serum. Serum obtained from CRIg-Fc–, CRIg(A)-Fc–, and control-Fc–treated mice was incubated at 37°C for 45 min, and the levels of C3a des Arg and C5a des Arg were measured by ELISA. (C) Systemic treatment with CRIg-Fc, but not control-Fc, reduces the presence of C3 fragments (green fluorescence), but not anti-collagen antibody (red fluorescence) on the cartilage surface in the metatarsal-phalangeal joints 14 d after antibody injection. (D) CRIg inhibits the production of C3a des Arg in the joints. Joints obtained at various time points after antibody injection were homogenized, and C3a des Arg was measured by ELISA. (E) CRIg-Fc and C3 fragments colocalize to the cartilage surface in the joints. Joints were obtained from mice 1 d after treatment with CRIg-Fc on day 5 after antibody injection. Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, CRIg-Fc versus control-Fc and CRIg(A)-Fc. Bar, 100 μm. A.U., arbritary units.

Article Snippet: C3a des Arg levels were quantified with capture and detection antibodies from BD Biosciences ( ) or with a mouse C3a des Arg ELISA kit (Bachem; ).

Techniques: Activation Assay, In Vitro, In Vivo, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Injection

Journal: Cell

Article Title: A mast cell receptor mediates post-stroke brain inflammation via a dural-brain axis

doi: 10.1016/j.cell.2025.06.045

Figure Lengend Snippet: (A) Representative flow cytometry gating of WT/ Mrgprb2 −/− brains 48h post-tMCAO. left , Live cells are gated using an immune marker (CD45) and myeloid marker (CD11b). right , Myeloid cells are gated using Ly6G and Ly6C to delineate CD45-high neutrophils and monocytes/macrophages, and CD45-low microglia. (B-D) (B) Absolute count of neutrophils, (C) monocytes/macrophages, and (D) CD11b-positive microglia in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mice 48h post-tMCAO (WT n =18, Mrgprb2 −/− n =16). (E) Representative immunofluorescence images of WT (left)/ Mrgprb2 −/− (right) right brain hemispheres 48h post-tMCAO. top row, CD45-positive immune cells. middle row, GFAP-positive activated astrocytes. Dotted white line separates infarcted tissue without GFAP and live, injured brain tissue with GFAP. bottom row, Merged channels with DAPI. Scale bar=50 μm. (F), left, CCL2 and right, CCL3 protein expression measured by ELISA in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mice (CCL2: WT n =6, Mrgprb2 −/− n =6, CCL3: WT n =5, Mrgprb2 −/− n =5). (G-H) (G) IL-6 and (H) neutrophil elastase protein expression measured by ELISA in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mice (G, WT n =5, Mrgprb2 −/− n =5, H, WT n =7, Mrgprb2 −/− n =7). (I) Representative immunofluorescence image of Mrgprb2 −/− mouse meninges 8 weeks after engraftment with Mrgprb2-tdT mast cells. left column, tdT denotes engrafted cells. middle column, Avidin denotes all mast cells. right column, Merged channels with DAPI. Scale bar=50 μm. (J) Mast cell count in meninges of Mrgprb2 −/− mice engrafted with saline, WT, or Mrgprb2 −/− mast cells, determined by Avidin-positive cells in the dura, using a 0.408mm 2 viewing frame (WT saline n =4, WT engraftment n =5, Mrgprb2 −/− saline n =4, Mrgprb2 −/− engraftment n =4). (K) Absolute count of neutrophils and monocytes/macrophages in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mast cell engrafted mice (WT n =16, Mrgprb2 −/− n =18). (L). left, Representative T2-weighted MRIs, and right , stroke volume of WT/ Mrgprb2 −/− MC-engrafted Mrgprb2 −/− mice 48h post-tMCAO (WT n =9, Mrgprb2 −/− n =8). Statistical analyses: Kruskal-Wallis test (B and G), two-way ANOVA with Sidak’s multiple comparisons test (C-D, F, H, and J-K), and two-sided Student’s t-test (L). Statistical test for C, D, and K was performed on log-transformed data to adjust for non-normality. Bar graphs indicate mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Mouse Semaphorin 3A ELISA Kit , Cusabio , Cat#: CSB-EL020980MO.

Techniques: Flow Cytometry, Marker, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Avidin-Biotin Assay, Cell Counting, Saline, Transformation Assay

Journal: Frontiers in immunology

Article Title: Anti-inflammatory actions of Pentosan polysulfate sodium in a mouse model of influenza virus A/PR8/34-induced pulmonary inflammation.

doi: 10.3389/fimmu.2023.1030879

Figure Lengend Snippet: FIGURE 5 Complement protein measurement in the lungs and serum of PR8-infected mice treated with PPS. (A) Changes in lung levels of C3a, C3b and C5a measured by ELISA. Values normalised to tissue weights. (B) Changes in serum concentration of C3a, C3b and C5a measured by ELISA. Box-whiskers plot show all data points (max/min), standard error, median (line in box) and mean (‘+’ in box). Statistically significant differences were assessed by one- way ANOVA with a Sidak post-test (ns, not significant);. Data shown is from three individual, independent experiments denoted Expt#1, Expt#2 and Expt#3 (for C3a in 5a, 5b; total n=12-18 mice) and two independent experiments denoted Expt#1 and Expt#2 (for C3b in (C, D) and C5a in (E, F) total n= 7-12 mice).

Article Snippet: Complement proteins were quantified using single ELISA kits (Novus Biologicals Mouse Complement C3a ELISA kit, Cat#NBP2-70037; Thermo Fisher Mouse Complement C5a ELISA kit, Cat#EHMC; Hycult Biotech Mouse Complement C3b ELISA kit, Cat#HK216-01; Biomatik mouse Terminal Complement Complex C5b9 (Cat#EKU10320); Cusabio mouse Factor B (Complement Factor Bb; CF-Bb) Cat#CSB-E04695m); R&D Systems mouse soluble ICAM-1 (sICAM-1) Quantikine Cat#MIC100) according to manufacturer’s instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in immunology

Article Title: Anti-inflammatory actions of Pentosan polysulfate sodium in a mouse model of influenza virus A/PR8/34-induced pulmonary inflammation.

doi: 10.3389/fimmu.2023.1030879

Figure Lengend Snippet: FIGURE 6 Pro-inflammatory cytokine/chemokine measurement in the lungs and serum of PR8-infected mice treated with PPS. Changes in lung and serum levels of CCL2, IL-6, IL-12p70, IL-10, IFN-g and TNF-a measured by ELISA in Experiment 1. Values for lung concentrations were normalised to tissue weights. Values for serum concentrations are shown as per mL of serum. Box-whiskers plot show all data points (max/min), standard error, median (line in box) and mean (‘+’ in box). Statistically significant differences were assessed by one-way ANOVA with a Sidak post-test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns: not significant). (n=6 mice per group per experiment; Data shown is one of 3 independent experiments).

Article Snippet: Complement proteins were quantified using single ELISA kits (Novus Biologicals Mouse Complement C3a ELISA kit, Cat#NBP2-70037; Thermo Fisher Mouse Complement C5a ELISA kit, Cat#EHMC; Hycult Biotech Mouse Complement C3b ELISA kit, Cat#HK216-01; Biomatik mouse Terminal Complement Complex C5b9 (Cat#EKU10320); Cusabio mouse Factor B (Complement Factor Bb; CF-Bb) Cat#CSB-E04695m); R&D Systems mouse soluble ICAM-1 (sICAM-1) Quantikine Cat#MIC100) according to manufacturer’s instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in molecular neuroscience

Article Title: Basolateral Amygdala Mediates Central Mechanosensory Feedback of Musculoskeletal System.

doi: 10.3389/fnmol.2022.834980

Figure Lengend Snippet: FIGURE 5 | Decreased Sema3A secretion with sensory nerve dysfunction in aging and muscle atrophic mice. (A) ELISA of serum Sema3A levels in mice at different ages, i.e., 7, 35, and 68 weeks old. (B) ELISA of serum Sema3A levels after BTxA-induced muscle dysfunction. (C) Chemogenetic activation of BLACaMKII neurons after muscle paralysis. (D) ELISA of serum Sema3A levels in BTxA model after chemogenetic activation of BLACaMKII neurons. (E) Representative immunofluorescent staining images of CGRP (green) in femurs of saline- or BTxA-treated mice. Scale bar, 50 µm. (F) Representative immunofluorescent staining images of CGRP (green) in tibia after chemogenetic manipulation of BLACaMKII neurons. Scale bar, 50 µm. (G) Quantitative analysis of CGRP+-nerve fibers in BTxA-injected mouse models. (H) Quantitative analysis of CGRP+-nerve fibers in hM3Dq vs. mCherry group of BTxA-injected mice. All data represent mean ± SD. Statistical analysis was assessed using student t-tests for panel (D) and one-way ANOVA followed Tukey comparison test for panels (A,B). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

Article Snippet: Human Sema3A s was measured with human Sema3A ELISA Kit (CUSABIO, CSB-E15913h), according to manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Staining, Saline, Injection, Comparison

Journal: Frontiers in molecular neuroscience

Article Title: Basolateral Amygdala Mediates Central Mechanosensory Feedback of Musculoskeletal System.

doi: 10.3389/fnmol.2022.834980

Figure Lengend Snippet: FIGURE 6 | Clinical cases for potential application of Sema3A as a prognostic signal of bone loss. (A) Correlation between BMD (T-score) and age (years) in 18 patients. (B) Correlation between BMD (T-score) and Sema3A level in 18 patients. (C) Serum Sema3A level between normal BMD group (T-score > –1) and low BMD group (T-score ≤–1). (D) Correlation between BMD (T-score) and Sema3A level in 18 patients. (E) Correlation between BMD (T-score) and Sema3A level in male group. (F) Correlation between BMD (T-score) and Sema3A level in female group. For all clinical data, linear regression models were used to explore the correlation between two variables. P < 0.05 was considered significant. (G) Basolateral amygdala (BLA) mediates mechanosensory role of bone in response to mechanical unloading.

Article Snippet: Human Sema3A s was measured with human Sema3A ELISA Kit (CUSABIO, CSB-E15913h), according to manufacturer’s instructions.

Techniques: