Journal: Scientific Reports

Article Title: Inflammatory transcriptomic signatures in a human cellular NMOSD model reveal upregulation of NF-κB and IL6 pathways

doi: 10.1038/s41598-025-27335-9

Figure Lengend Snippet: Complement component deposition on the surface of different cell lines after application of the monoclonal AQP4 antibody E5415A (10 µg/mL) and active or heat-inactivated human complement. ( A ) Terminal complement complex (TCC) formation on U-87MG-AQP4-ECFP cells. After the treatments, cells were washed with PBS with 10% heat-inactivated FCS, and mouse anti-human C5b-C9 neo (aE11) AlexaFluor594 (Novus Biologicals) was applied with 5 µg/mL to visualize TCC deposition on the cell surface. ( B–D ) Complement component C3/C3b/iC3b deposition. After the treatments, cells were washed with PBS with 10% heat-inactivated FCS, and mouse anti-human/mouse C3/C3b/iC3b purified (6C9) (Cedarlane) was applied with 5 µg/mL, followed by 2 µg/mL goat-anti mouse IgG1 Cross-Adsorbed Secondary Antibody, AlexaFluor594 (Invitrogen, Thermo Fisher Scientific) to visualize opsonization by C3/C3b/iC3b on the cell surface of ( B ) U-87MG-AQP4-ECFP cells, ( C ) U-87MG-ECFP cells and ( D ) primary human astrocytes. Scale bar = 20 μm. AF , Alexa Fluor; AQP4, aquaporin-4; DAPI, 4′,6-diamidino-2-phenylindole; ECFP, enhanced cyan fluorescent protein.

Article Snippet: After the treatments, cells were washed with PBS with 10% heat-inactivated FCS, and mouse anti-human C5b-C9 neo (aE11) AlexaFluor594 (Novus Biologicals) was applied with 5 μg/mL to visualize TCC deposition on the cell surface. ( B–D ) Complement component C3/C3b/iC3b deposition.

Techniques: Purification

Journal: Scientific Reports

Article Title: Inflammatory transcriptomic signatures in a human cellular NMOSD model reveal upregulation of NF-κB and IL6 pathways

doi: 10.1038/s41598-025-27335-9

Figure Lengend Snippet: Immunocytochemistry of U-87MG-AQP4-ECFP cells after treatment with NMOSD patient sera and human complement. U-87MG-AQP4-ECFP cells were treated with 10% NMOSD patient sera (with or without AQP4-IgG) in combination with active or heat-inactivated human complement. Then, the cells were washed with PBS with 10% heat-inactivated fetal calf serum, and immunocytochemistry was performed. ( A ) Terminal complement complex (TCC) staining. The cellular surface was stained for TCC deposition by applying mouse anti-C5b-C9 neo (aE11) AlexaFluor594 (Novus Biologicals) with 5 µg/mL. ( B ) NF-κB component p65 staining. Translocation of p65 from the cytosol into the nucleus was visualized by intracellular immunocytochemistry. Cells were fixed, permeabilized, and blocked. Then, 2 µg/mL rabbit anti-human p65 (D14E12) (Cell Signaling) served as the primary antibody, followed by goat anti-rabbit AlexaFluor647 (Invitrogen, Thermo Fisher Scientific). White arrows indicate p65 translocation into the nucleus. Scale bar = 20 μm. AF, Alexa Fluor; DAPI, 4′,6-diamidino-2-phenylindole; TCC, terminal complement complex.

Article Snippet: After the treatments, cells were washed with PBS with 10% heat-inactivated FCS, and mouse anti-human C5b-C9 neo (aE11) AlexaFluor594 (Novus Biologicals) was applied with 5 μg/mL to visualize TCC deposition on the cell surface. ( B–D ) Complement component C3/C3b/iC3b deposition.

Techniques: Immunocytochemistry, Staining, Translocation Assay

Journal: Scientific Reports

Article Title: Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood

doi: 10.1038/s41598-017-04898-w

Figure Lengend Snippet: ( A–C ) CHC coating of PAEC reduces complement deposition, cellular activation and coagulation induced by human plasma. Uncoated and CHC-coated PAEC were treated for 4 hrs with 10% human plasma, and assessed by ELISA for ( A ) the deposition of activated complement C5b-9 and ( B ) the expression of the adhesion molecule E-selectin ( B ). ( C ) Supernatants from this assay were measured for D-dimers concentration. CHC coating significantly protected WT PAEC. Uncoated GTKO.hCD46.hTBM PAEC were significantly protected compared to uncoated WT PAEC; coating with CHC further reduced complement deposition and cellular activation, although this was not statistically significant. ( D,F ) CHC coating of hTNFα-activated PAEC reduces complement deposition, cellular activation and coagulation induced by human plasma. WT and GTKO.hCD46.hTBM PAEC were treated with hTNFα (100 ng/ml) for 2 hrs to induce cellular activation and shedding of the endothelial glycocalyx. After, PAEC were coated with CHC and incubated for 4 hrs with 10% human plasma, and assessed for ( D ) complement C5b-9 deposition and ( E ) endothelial E-selectin expression as well as ( F ) D-dimer levels in the supernatants. CHC coating significantly protected hTNFα-activated WT and GTKO.hCD46.hTBM PAEC compared to uncoated PAEC. Significance was measured by one-way ANOVA with Bonferroni correction (*p < 0.05, **p < 0.01, ***p < 0.001). Data are mean ± SD of three independent experiments.

Article Snippet: Rabbit anti-human C3b/c FITC (A0062; Dako), mouse anti-human C5b-9 FITC (clone aE11; HM2167F, Hycult Biotech), and mouse anti-human CD62E FITC (clone 1.2B6, MCA883F, AbD Serotec) were diluted in PBS/1% BSA and incubated for 60 min at RT followed by three washes.

Techniques: Activation Assay, Coagulation, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Incubation