Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

doi: 10.1111/j.1538-7836.2006.02033.x

Figure Lengend Snippet: Fig. 4. Complement C3 deposition in joints. Healthy 5–8-week old female TMwt/wt (A) and TMLeD/LeD (B) mice were killed and knee joints were processed for immunohistochemical staining to detect complement factor C3 deposition. These sections are representative of three mice. No C3 was detectable in the joints of TMwt/wt mice (A). C3 was readily observed on the articular surface (small arrows) and in the bone marrow space of TMLeD/LeD mice (large arrows) (B). Higher power views of the articular surface are shown in the right upper corner of each panel.

Article Snippet: Specific immunostaining of histologic sections was achieved by overnight incubation with the following primary antibodies: CD45 (rat, 1:100; BecktonDickinson, Erembodegem, Belgium, no. 553076);Mac3 (rat, 1:300; BecktonDickinson, no. 553322); MPO (rabbit, 1:100, Dako A/S, Glostrup, Denmark, no. A0398); HMGB1 (rabbit; 1:250, Santa Cruz Biotechnology, Boechout, Belgium, no. sc12523); and complement factor C3 (rat, 1:200; Cedarlane,Uden, theNetherlands, no. CL7503AP), which recognizes intact C3, C3b, iC3b and C3d, but not C3a.

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Complement is fixed on KEL RBCs in-vivo and in-vitro in response to polyclonal anti-KEL (KELIg). (A) General in-vivo experimental design: recipients were treated with or without KELIg and transfused with DiO labeled KEL RBCs. (B) C3, C4, and Factor B were measured on recovered DiO positive KEL RBCs 1-hour post-transfusion; black filled histogram shows condition without KELIg; grey shaded histogram shows condition with KELIg. (C) In-vitro experiments were completed with KELIg incubated with KEL RBCs in the presence (darkest histogram) or absence (lightest histogram) of serum; an additional condition included saline incubated with KEL RBCs in the presence of serum (medium grey histogram). (D) Additional in-vitro conditions included KELIg incubated with KEL RBCs in the presence of serum (darkest histogram), in the presence of serum with EGTA-Mg (lightest histogram) or serum with EDTA (medium grey histogram). These data are representative of 3 independent experiments; p < 0.05 for C3 detection on RBCs in the presence or absence of KELIg.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vivo, In Vitro, Labeling, Incubation, Saline

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Recipient C3 and C1q contribute to KELIg immunoprophylaxis failure in the setting of poly (I:C). (A) General in-vivo experimental design: recipients were infused with KELIg and transfused the following day in the absence or presence of poly (I:C) treatment, with anti-KEL responses evaluated longitudinally. (B) Anti-KEL IgG responses in wild type compared with C3 -/- recipients transfused in the absence of poly (I:C). (C) Anti-KEL IgG responses in wild type compared with C3 -/- recipients transfused in the presence of poly (I:C). (D) Anti-KEL IgG responses in wild type compared with C1q -/- recipients transfused in the presence of poly (I:C). (E) Baseline impact of recipient treatment with poly (I:C) on alloimmune responses to KEL RBCs in the absence of KELIg. These data are representative of 2-3 independent experiments, with 3-5 mice/group/experiment; error bars indicate standard deviation between individual mice. *p < 0.05 (C, D) d14,21,28, and all comparisons for (E) .

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vivo, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Recipient complement, KEL RBC clearance, and KEL antigen expression. KEL RBCs were labeled with DiO and mixed with wild type RBCs labeled with DiI; this mixture was transfused into recipients that had been infused with KELIg and treated with poly (I:C). (A) shows wild type compared with C3 -/- recipients; (B) shows wild type compared with C1q -/- recipients. Recovered DiO labeled RBCs were then evaluated for KEL glycoprotein expression by flow cytometry, after incubation with KELIg and fluorescently conjugated anti-mouse IgG; (C) shows wild type recipients compared with C3 -/- recipients; (D) shows wild type recipients compared with C1q -/- recipients. These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice. *p < 0.05 for all comparisons in (C) and p = ns, not significant for all comparisons in (D) .

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Expressing, Labeling, Flow Cytometry, Incubation, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Poly (I:C) and KELIg immunoprophylaxis result in increased transfused KEL RBC consumption by splenic inflammatory monocytes in wild type compared with C3 -/- mice. DiO labeled KEL RBCs were transfused to wild type or C3 -/- mice treated with KELIg in the presence or absence of poly (I:C) and splenic cell subsets were evaluated at 1 and 16 hours post-transfusion (A, B) . (C) Representative histograms for DiO RBC fluorescence patterns of inflammatory monocytes at 16 hours post-transfusion, after first excluding TER119 positive RBCs on the exterior of the splenic cells; black open histogram is KELIg in wild type, dotted open histogram is KELIg and PIC in wild type; white shaded histogram is KELIg in C3 -/- and dotted shaded histogram is KELIg and PIC in C3 -/- . DiO mean fluorescence intensity (MFI) of the splenic cell subsets was evaluated at 1 hour (D) and 16 hours (E) post-transfusion. These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Labeling, Fluorescence, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: Complement-fixed KEL RBCs bind in-vitro to wild type donor derived B-cells but not to CR1/2 -/- B-cells. DiO labeled KEL RBCs were incubated with KELIg in the presence of sera, followed by incubation with peripheral blood derived WBCs from donor mice. (A) In wells using WBCs from wild type donor mice, CD19+B220+ B-cells cells were separated by DiO positivity and then by C3 positivity. The CD19+B220+ cells were next separated by their CD23 and CD21/35 expression, with the cells highest for CD23 and CD21/35 gated in the larger gate and those less strongly positive for CD23 and CD21/35 gated in the smaller gate; the DiO positive and negative populations were then evaluated for their C3 positivity. Shaded histograms are the DiO positive population, open histograms are the DiO negative population. (B) In wells using WBCs from CR1/2 -/- mice, CD19+B220+ B-cells cells were separated by DiO positivity and then by C3 positivity. Shaded histograms are the DiO positive population, open histograms are the DiO negative population. These data are representative of more than 3 independent experiments, with 2 involving CD23 and CD21/35 staining.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: In Vitro, Derivative Assay, Labeling, Incubation, Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

doi: 10.3389/fimmu.2021.704072

Figure Lengend Snippet: CR1/2 are required for KELIg immunoprophylaxis failure in the setting of poly (I:C). (A) KEL RBCs were transfused into wild type recipients or recipients lacking CR1/2, in the presence or absence of poly (I:C). (B) RBCs were labeled with DiO and mixed with wild type RBCs labeled with DiI; this mixture was transfused into wild type or CR1/2 -/- recipients that had been infused with KELIg and treated with poly (I:C). Recovered DiO positive KEL RBCs were evaluated for (C) KEL glycoprotein expression, and (D) bound complement C3. (E) Wild type or CR1/2 -/- recipients were infused with KELIg and transfused the following day in the absence or presence of poly (I:C), with anti-KEL responses evaluated longitudinally. *p < 0.05 for d14, 21 and 28 of (A) between CR1/2 -/- mice treated with or without poly (I:C); p < 0.05 for 10 min and 1-hour timepoints in (C) ; p < 0.05 for d14, 21, and 28 in (E) . These data are representative of 2-3 independent experiments with 3 mice/group/experiment; error bars indicate standard deviation between mice. ns, not significant.

Article Snippet: Following transfusion of KEL RBCs, complement was evaluated on the visualized RBC surface using antibodies against C3 (Cl7503B, Cedarlane, Ontario, Canada), C4 (clone 16D2, Santa Cruz), or Factor B (CL8824AP, Cedarlane, Ontario, Canada) followed by streptavidin or a fluorescently conjugated donkey anti-rabbit antibody (BioLegend, San Diego, California).

Techniques: Labeling, Expressing, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV

doi: 10.1111/j.1582-4934.2012.01648.x

Figure Lengend Snippet: Immunohistochemical staining for Col IV NC1 isoforms. (A) Col IV NC1 domains α1–α6 were stained in human lung tissue sections using specific antibodies and detected using a secondary antibody conjugated to fluorescein isothiocyanate (FITC) (green staining). Eight lymphangioleiomyomatosis (LAM) patients and 10 controls were tested. Scale bar represents 50 μm. L: lumen; B: basal lamina; V: blood vessel. (B) Col IV α3 (red staining) and α5 (green staining) NC1 domains were detected in human lung tissue sections from four cystic fibrosis patients and one bronchiectasis patient. The isotype control was exposed to rat IgG and the secondary antibody.

Article Snippet: Sections were then washed with PBS and a goat anti-rat fluorescein isothiocyanate (FITC) [MP Biomedicals, Solon, OH, USA (1 ng/ml)] conjugated secondary antibody was added, and incubated for 30 min. at room temperature.

Techniques: Immunohistochemical staining, Staining

Journal: Medical Microbiology and Immunology

Article Title: Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes : understanding the role of complement properdin

doi: 10.1007/s00430-013-0324-z

Figure Lengend Snippet: Complement activation, phagocytosis and intravascular survival of S. pneumoniae D39. a C3 reactivity of separated bacterial pellets after incubation with mouse sera (pool n = 6) under conditions favouring the alternative pathway (GVB ++ /EGTA/Mg 2+ ) or classical pathway (GVB ++ ) activation, 37 °C, 30 min (Western blot); lane 5 shows specificity of rat antimouse C3 antibody (only D39, no serum). b Pools of 9 sera-matching each cell genotype were used to opsonise FITC-labelled S. pneumoniae before addition to respective mouse splenocytes for 1 h. Flow cytometry was performed, while quenching extracellular signal with trypan blue (representative analysis of n = 2). C After 24 h’ infection of splenic macrophages with D39, there is decreased TNF-α production (measured using L929 assay) by splenocytes from properdin-deficient mice in the presence of properdin-deficient sera compared to wildtype cells with wildtype sera. d Both genotypes were injected i.v. with 10e6 D39 and bacterial counts (±SEM) determined for each of 3 days from n = 5 each genotype ( p < 0.05)

Article Snippet: Lung homogenates from control and infected mice were analysed by Western blot using HRP-conjugated goat antimouse C3 (ICN pharmaceuticals Inc.), and the density of bands corresponding to the C3 activation products was measured.

Techniques: Activation Assay, Incubation, Western Blot, Flow Cytometry, Infection, Injection