Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: All inductions were performed for 3 days with 1 ×10 6 CD62L hi Foxp3 − CD25 − CD4 + T cells, rhIL-2 (5 ng/ml), and anti-CD3 + CD28 beads (per manufacturer’s instructions), experiments were repeated two times. ( a ) WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 − cells were activated and assayed for percent Foxp3 + CD25 + by flow cytometry (*P<0.001, n=5). (b) Following iT reg induction (as in a ), flow sorted WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were incubated with 10 6 CellTracker Red™ labeled CD25 − Foxp3 − CD4 + WT cells in differing T eff /iT reg ratios + anti-CD3 (5 μg/ml) and 2.5×10 5 CD11c + DCs. Relative suppressive capacity was determined by percent Red-labeled dividers (n=5). (c) WT Foxp3 − CD4 + T cells were induced as in (a) in the absence or presence of anti-C3a (10 μg/ml), anti-C5a (10 μg/ml), or both or in the absence and presence of the antagonists C3aR-A (10 nM), C5aR-A (10 nM), or both. CD4 + T cells then were assayed for Foxp3 expression by flow cytometry (*P<0.001, n=6). (d) Following iT reg induction (as in a ), sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were washed and recultured for 24 hr in the absence of further stimulation. Culture supernatants were assayed for TGF-β, IL-6, and IL-10 by ELISA (*P<0.001, n=6). (e) C3ar1 −/− C5ar1 −/− iT regs were induced (as in a) in the absence and presence of anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Foxp3 + CD25 + T reg percentages were quantified by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Flow Cytometry, Incubation, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: iT reg induction and activation experiments were performed as in with 2.5×10 5 CD11c + DCs and anti-CD3 mAb (5 μg/ml) instead of anti-CD3+CD28 beads, experiments were repeated two times. (a) Sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with WT DCs ± TGF-β1 (5 ng/ml) and assayed for CD25 and Foxp3 expression by flow cytometry (*P<0.001, n=10). (b) (Left) WT Foxp3 − CD4 + T cells were incubated with C3 −/− C5 −/− DCs without TGF-β1 ± C3a/C5a (100 ng/ml) and Foxp3 + CD25 + cells quantified by flow cytometry. (Right) WT Foxp3 − CD4 + T cells were incubated in the presence of TGF-β1 (5 ng/ml) ± C3a/C5a (100 ng/ml) and percent Foxp3 + CD25 + cells quantified (*P<0.001, n=7). (c) Sorted WT or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with C3ar1 −/− C5ar1 −/− DCs in the absence or presence of C5a (100 ng/ml) after which percent Foxp3 + CD25 + cells was quantified. The % Foxp3 + cells with DKO or WT T cells did not significantly differ (P=0.54). (d) Sorted C3 −/− C5 −/− or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with Daf1 −/− DCs ± anti-C3a and anti-C5a mAbs after which percent Foxp3 + CD25 + cells was quantified (*P<0.001; n=6). (e) Sorted WT Foxp3 − or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated for 24 hr either in the absence of DCs or in the presence of WT DCs or C3ar1 −/− C5ar1 −/− DCs. Supernatants then were assayed for TGF-β1 and IL-6 by ELISA (*P<0.001, n=6). (f) C3ar1 −/− C5ar1 −/− Foxp3 − cells were incubated for 3 days with anti-CD3 (5 μg/ml), IL-2 (5 ng/ml), and WT DCs ± anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Following the 3 day induction, Foxp3 + CD25 + T reg percentages were determined by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) WT CD4 + T cells were incubated for 30 min with anti-CD3+CD28 beads after which they were incubated for 10 min with Forskolin (30 μM) ± C3a (100 nM), C5a (100 nM), or both. Levels of cAMP activity were determined by cAMP-Glo assay (*P<0.001). (b) WT, C3ar1 −/− , C5ar1 −/− , and C3ar1 −/− C5ar1 −/− CD4 + T cells were stimulated with anti-CD3 mAb for 30 min after which PKA activity was quantified by PepTag assay. (c) iT regs generated from sorted WT Foxp3 − CD4 + T cells plus TGF-β1, iT regs derived from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells, and conventional Foxp3 − CD4 + T cells were stimulated with anti-CD3+CD28 beads for 15 min and assayed for S 473 and T 308 p-AKT by Luminex assay (n=5). (d and e) WT or C3ar1 −/− C5ar1 −/− CD4 + T cells were incubated for 15 min ± anti-CD3+CD28 beads. Cells were extracted in phospho-lysis buffer and extracts assayed for (d) phospho-rbS6 and (e) phospho-Smad2 by immunoblotting. (f) iT regs induced from WT Foxp3 − CD4 + T cells plus TGF-β1 or from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were assayed for phospho-STAT3 and phospho-STAT5 by flow cytometry (representative plots of n=6).

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Activity Assay, Glo Assay, Generated, Derivative Assay, Luminex, Lysis, Western Blot, Flow Cytometry

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Sorted WT Foxp3 − cells were incubated for 1 hr with anti-CD3+CD28 beads (as in ) in the absence or presence of TGF-β1 (5 ng/ml) and then assayed for complement mRNA transcripts by qPCR (*P<0.001, n=5). (b) Sorted WT Foxp3 − CD4 + T cells were incubated for 1 hr with anti-CD3+CD28 beads plus TGF-β1 (5 ng/ml) alone, TGF-β1 (5 ng/ml) plus IL-6 (5 ng/ml), or IL-6 (5 ng/ml) alone, after which the cells were assayed for C3 mRNA expression by qPCR (*P<0.001, **P<0.02, n=5). (c and d) Sorted WT Foxp3 − CD4 + T cells were incubated for 48 hr with anti-CD3+CD28 beads plus TGF-β1 alone, TGF-β1+IL-6, or IL-6 alone as in (b) . Culture supernatants were assayed for (c) C3a and C5a generation by ELISAs, and (d) C3aR and C5aR surface expression by flow cytometry (P<0.01 for TGF-β1 alone vs TGF-β1+IL-6 or IL-6 alone, Mean Fluorescence Intensity (MFI) values; n=5). (e) Sorted WT Foxp3 − cells were activated for 48 hr with anti-CD3+CD28 beads ± C5a (100 ng/ml) or C3aR-A/C5aR-A (10 nM), after which culture supernatants were assayed for TGF-β1 or IL-6 by ELISA (P<0.05; n=6). (f) Sorted WT Foxp3 − CD4 + T cells were incubated with WT DCs or with C3 −/− C5 −/− DCs in the absence of TGF-β1 ( C3 −/− C5 −/− iT regs ). DCs (left side) were assayed for C5aR and C5L2 expression by gating on CD11c + cells. Responder T cells (right side) were assayed for C5aR and C5L2 expression by gating on Foxp3 − cells in the case of WT CD4 + T cells prepared with WT DCs and on Foxp3 + cells in the case of WT CD4 + prepared with C3 −/− C5 −/− DCs. (*P < 0.01; n=5). (g) Sorted WT or C5L2 −/− (encoded by Gpr77) Foxp3 − CD4 + T cells were incubated with WT DCs (Left) and sorted WT Foxp3 − CD4 + T cells were incubated with WT or C5L2 −/− DCs (right) both in the presence of anti-CD3 and TGF-β1 (5 ng/ml). Percent Foxp3 + CD25 + cells were assayed by flow cytometry. (*P<0.001, n=6). (h) WT Foxp3 − CD4 + T cells were incubated for 3 days with WT DCs plus TGF-β1 (5 ng/ml) after which Foxp3 + and Foxp3 − cells were sorted. Foxp3 − CD4 + T cells or Foxp3 + CD4 + iT regs were incubated for 5 min with anti-CD3+CD28 beads + biotin-labeled C5a, the cells chilled to 4°C, and plasma membrane fractions were isolated by ultracentrifugation. Following C5aR and C5L2 IP, bound proteins were eluted with alkaline solution, and equal amounts of protein (determined by A 280 ) were loaded onto SDS PAGE gels. Following electrophoresis, gels were blotted for biotinylated-C5a by adding streptavidin-HRP followed by ECL reagent.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Labeling, Membrane, Isolation, SDS Page, Electrophoresis

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Flow sorted human CD45RA + CD25 − CD4 + T cells (1×10 6 ) were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous CD11c + DCs in the absence or presence of 1) TGF-β1 (5 ng/ml), 2) each of C3aR-A/C5aR-A (10 nM), or 3) each of anti-C3a/C5a mAbs (10 μg/ml). Percent Foxp3 + CD25 + CD4 + T cells then were determined by flow cytometry. (b) Flow sorted CD25 + cells from (a) were incubated for 3 days with in differing T eff /iT reg ratios with 1×10 6 CFSE labeled autologous naive CD25 − cells, anti-CD3 mAb (3 μg/ml), and 1×10 4 autologous CD11c + DCs. Percent dividers was determined by CFSE dilution. (c) CD45RA + CD25 − CD4 + T cells (1×10 6 ) were isolated from 5 healthy controls (NC) and 3 MS patients by flow sorting. The cells were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous DCs in the absence or presence of 1) rhTGF-β1 (5 ng/ml), 2) C3aR-A/C5aR-A (10 nM), or 3) anti-C3a/C5a mAbs (5 μg/ml). Cells were washed and sorted on CD25. After sorting, CD25 + (T reg ) were incubated for 3 days with anti-CD3 mAb, 2.5×10 5 autologous DCs, and 10 6 CD25 − (Effector) cells prelabeled with CFSE. Percent dividers was determined by CFSE dilution.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Flow Cytometry, Labeling, Isolation

Journal: Computational and Structural Biotechnology Journal

Article Title: The role of microglia and complement C5/C5a in the pathogenesis of rhegmatogenous retinal detachment with choroidal detachment

doi: 10.1016/j.csbj.2025.08.019

Figure Lengend Snippet: scRNA-seq Analysis of Complement C5 Components and Intercellular Communication in Retinal Detachment. (A) UMAP feature plots (left) show C5, C5AR1, and C5AR2 expression across retinal cell clusters in RRD and RRDCD, with color intensity indicating expression levels. Violin plots (right) compare cell type-specific expression distributions between conditions. (B) CellPhoneDB-derived dot plot displays ligand-receptor interactions, with bubble size reflecting interaction significance (-log₁₀ p-value) and color denoting mean expression. Comparisons include C5-C5AR1, C3-C5AR2, TNF-TNFRSF1A/B, and VEGF (VEGFA-FLT1, VEGFB-NRP1) pathways between RRD and RRDCD.

Article Snippet: Experimental groups were treated with complement C5 (HY-P7695; MedChemExpress) at concentrations of 0.5 μg/mL, 1.0 μg/mL, and 2.5 μg/mL, while the control group received no complement C5.

Techniques: Expressing, Derivative Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: The role of microglia and complement C5/C5a in the pathogenesis of rhegmatogenous retinal detachment with choroidal detachment

doi: 10.1016/j.csbj.2025.08.019

Figure Lengend Snippet: Characterization of complement C5 effects on cellular responses and molecular markers. (A) MTT assay assessing the viability/metabolic activity of RF/6 A choroidal vascular endothelial cells after 24-hour co-culture with primary mouse retinal microglia that were pre-treated with varying concentrations of complement C5 (0, 0.5, 1.0, 2.5 µg/mL)， with results expressed as ODsample/ODblank ratios. (B) Apoptotic effects of C5-treated (1 μg/mL, 24 h) microglia on RF/6 A choroidal vascular endothelial cells, as evidenced by TUNEL staining (red) and quantification of TUNEL-positive area. (C) C5 exposure (1 μg/mL, 24 h) altered microglial cell viability (MTT assay, Student's t -test) and morphology (brightfield images). (D) ZO-1 immunofluorescence (red) in ARPE-19 cells co-cultured with C5-treated retinal microglia (1 μg/mL, 24 h) showed tight junction integrity (nuclei: DAPI, blue). (E) CD86 expression (red) increased in retinal primary mouse microglia following C5 treatment (1 μg/mL, 24 h). (F) Western blot confirmed elevated CD86 protein levels in C5-treated microglia (1 μg/mL, 24 h). (G) ELISA detected significantly higher TNF-α and IL-1β secretion in C5-treated microglia (1 μg/mL, 24 h). Data represent mean ± SD of ≥ 3 independent experiments.

Article Snippet: Experimental groups were treated with complement C5 (HY-P7695; MedChemExpress) at concentrations of 0.5 μg/mL, 1.0 μg/mL, and 2.5 μg/mL, while the control group received no complement C5.

Techniques: MTT Assay, Activity Assay, Co-Culture Assay, TUNEL Assay, Staining, Immunofluorescence, Cell Culture, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors on iLite ® C5a Assay Ready Cells, displaying commercial mAb and small-molecule C5aR1 inhibitors ( a ) and in-house C5aR1 mAbs ( b ). Data were normalized to a stimulation control (added C5a) and analyzed by nonlinear curve fitting. Data points were collected in duplicates across multiple assay plates; each assay plate was repeated three times. Data are presented as mean ± SD.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Control

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors within a C5a-driven PMN calcium flux assay. A negative inhibition control (IgG1k isotype mAb) and a positive inhibition control (20 μ m avacopan) were included. After first recording the baseline, either C5a or buffer was added, followed by the addition of either ionomycin or buffer. Data are presented as fold increase based on the initially recorded baseline (mean FITC detector MFI). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were obtained in singlets. Data are presented as mean + SD (one-sided) for better visualization.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Calcium Flux Assay, Inhibition, Control

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors on C5a-stimulated PMNs ( a–c ), followed by titration of mAb 18-41-6-based F(ab’) 2 fragments versus equimolar concentrations of avacopan ( d , e ). a–c A negative control for C5aR1 inhibition (IgG1k isotype mAb) and a positive control (75 μ m avacopan) were included. Data points were normalized to a stimulation control sample (T10stim) with added C5a. a A baseline control (without incubation and without stimulation, Tb) and an incubation control (incubation and without stimulation, T10) are depicted. Titration curves of mAb 18-41-6-based F(ab’) 2 fragments and avacopan were analyzed by nonlinear curve fitting. The experiments were repeated three times with the blood of two different anonymous healthy donors ( a–c ) or one different anonymous healthy donor per repeat ( d , e ); data points were acquired in singlets. Data are presented as mean ± SD.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Titration, Negative Control, Inhibition, Positive Control, Control, Incubation

Journal:

Article Title: Site-specific CPB1 tyrosine nitration and patho-physiological implications following its physical association with NOS-3 in experimental sepsis

doi: 10.4049/jimmunol.0900593

Figure Lengend Snippet: CPB1 tyrosine nitration results in C5a accumulation in the mouse spleen following LPS administration. A) Spleen proteins from sham, LPS, LPS + NOS-2 inhibitor 1400W, LPS + L-NIO, LPS + Vinyl-L-NIO (relatively specific NOS-3 inhibitors), and LPS + Allopurinol (XO inhibitor) were separated by SDS-PAGE and electroblotted on a nitrocellulose membrane. Western blot analysis was performed using an antibody specific to C5a. B) A bar graph represents the normalized relative band intensities of the western analysis of the proteins immunoreactive to C5a antibody. C) Spleen proteins from sham (wild type), LPS (wild type), sham (NOS-3 knockout) and LPS (NOS-3 knockout) were separated by SDS-PAGE and electroblotted on a nitrocellulose membrane. Western blot analysis was performed using an antibody specific to C5a. D) A bar graph represents the normalized relative band intensities of the western analysis of the proteins immunoreactive to C5a antibody.* P<0.05 when compared to the sham-treated of wild type mice; #P<0.05 when compared to the sham-treated of knockout mice. E) Chemotaxis of HL 60 cells in response to recombinant C5a. Also, recombinant C5a was incubated with both mouse recombinant CPB1 and nitro-CPB1 to assess the effect of nitration of CPB1 on C5a-induced chemotaxis. * P<0.05 when compared to rC5a alone; #P<0.05 when compared to rC5a + mouse rCPB1. F) Chemotaxis of HL-60 cells in response to immunoprecipitated C5a from LPS-treated (wild type), XO inhibitor-treated, NOS-3 inhibitors treated, NOS-2 inhibitor-treated and LPS-treated knockout mice spleen tissue homogenates was measured in terms of relative fluorescent intensity by a CytoselectTM 96 well cell migration assay following the manufacturer’s protocol. . * P<0.05 when compared to sham-treated mice alone; #P<0.05 when compared to LPS-treated wild type mice.

Article Snippet: Mouse recombinant C5a (R&D Chemicals) was used as a positive control and incubated in the feeder layer.

Techniques: Nitration, SDS Page, Western Blot, Knock-Out, Chemotaxis Assay, Recombinant, Incubation, Immunoprecipitation, Cell Migration Assay

Journal:

Article Title: Site-specific CPB1 tyrosine nitration and patho-physiological implications following its physical association with NOS-3 in experimental sepsis

doi: 10.4049/jimmunol.0900593

Figure Lengend Snippet: Tyrosine nitration of CPB1 in the sinus lining cells of the spleen. Proximal association and binding of CPB1 and NOS-3 possibly resulted in higher nitration yield in the local milieu, leading to loss of CPB1 activity. This would possibly lead to accumulation of inflammatory mediators like C5a in the spleen, thus amplifying the systemic inflammatory response and altering immune pathology.

Article Snippet: Mouse recombinant C5a (R&D Chemicals) was used as a positive control and incubated in the feeder layer.

Techniques: Nitration, Binding Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Expression of Gα subunits in macrophages and roles of Gnai2 and Gnai3 in complement C5a-mediated chemotaxis. A, expression levels of Gα subunits in mouse resident peritoneal F4/80+ cells (macrophages). RNA-Seq analysis was performed using RNA isolated from resident peritoneal F4/80+ cells purified by cell sorting (n = 3 mice). Inset (superimposed graph with an interrupted y axis), expression levels of receptors for complement components 3a and 5a. Error bars, S.E. B, schematic diagram showing C5aR, a member of the G protein–coupled receptor superfamily, and a heterotrimeric G protein in which the subunits are color-coded blue (Gα), green (Gβ), and white (Gγ). The four Gα families (Gαi/o, Gαs, Gαq/11, and Gα12/13) are listed below the blue α-subunit (Gα) together with the names of the corresponding genes investigated with knockout mouse models, including two genes encoding β-subunits: Gnb1 (Gβ1) and Gnb2 (Gβ2). C, migration plots of WT, PTX-treated WT, Gnai2−/−, and Gnai3−/− macrophages in a chemotactic complement C5a gradient. D, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index), calculated by dividing the displacement along the y axis by the cumulative distance migrated. The chemotaxis index is also known as the y-forward migration index and has a range of −1 to +1. *, p < 0.05; Kruskal–Wallis test and post hoc Mann–Whitney U test with Bonferroni correction (n = 75 for each group, except n = 50 for the WT + PTX group; three independent experiments, except two independent experiments for the WT + PTX group).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Expressing, Chemotaxis Assay, RNA Sequencing, Isolation, Purification, FACS, Knock-Out, Migration, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Complement C5a-induced Ca2+ transients and lamellipodial membrane protrusions are not impaired in Gnai2−/− or Gnai3−/− macrophages. A, simultaneous imaging of intracellular [Ca2+] (green trace) and projected cell area (black trace) in individual WT and Gnai2−/− macrophages challenged with 20 nm complement C5a. Intracellular [Ca2+] is indexed as relative Cal-510 fluorescence intensity (F/F0) in which the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, summary box plots of peak complement C5a-induced Ca2+ transients and projected cell area. *, p < 0.05; n.s., not significant; Kruskal–Wallis test and post hoc Mann–Whitney U test with Bonferroni correction (n = 50 (WT), n = 19 (WT + PTX), n = 46 (Gnai2−/−), and n = 9 (Gnai3−/−); 2–3 independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Membrane, Imaging, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Sequestration of intracellular [Ca2+] with EGTA does not prevent complement C5a-induced lamellipodial membrane protrusions. A, example (green trace) of a complement C5a-induced Ca2+ transient largely blocked in a WT macrophage after passively loading the cell with the Ca2+ chelator EGTA using its AM ester form (EGTA/AM). The box plots on the right show peak complement C5a-induced Ca2+ transients measured in the absence and presence of EGTA/AM. B, the trace shows the projected cell area corresponding to the above Ca2+ trace (A). The box plots on the right show peak complement C5a-induced cell spreading in the absence and presence of EGTA/AM. *, p < 0.05; n.s., not significant; Mann–Whitney U test (n = 50 (WT pool) and n = 43 (WT + EGTA/AM); n = 3 independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Membrane, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Complement C5a-mediated chemotaxis is preserved in Gnaq/Gna11 double knockout and Gna12/Gna13 double knockout macrophages. A, schematic diagram highlighting genes of the Gαq/Gα11 (Gnaq and Gna11) and Gα12/Gα13 (Gna12 and Gna13) families of Gα subunits that potentially may be activated by C5aR. B, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index). *p < 0.05; Kruskal–Wallis test and post-hoc Mann–Whitney U test with Bonferroni correction (n = 75 for each group; 3 independent experiments). C, migration plots of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages in a chemotactic complement C5a gradient. D, 200 × 300-μm snapshots of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages in a chemotactic complement C5a gradient. Black arrows, elongated trailing ends. The schematic diagram on the left shows a μ-Slide Chemotaxis chamber with one of the two 40-μl reservoirs (filled with a blue dotted pattern) containing 20 nm complement C5a. E, box plots of maximal tail lengths developed by macrophages migrating in a chemotactic complement C5a gradient over a 6-h period. *, p < 0.05; Kruskal–Wallis test and post hoc Mann–Whitney U test with Bonferroni correction (n = 50 cells/group; sampled from two independent experiments). F, representative example, from two independent experiments, of RhoA activity measured using a colorimetric G-LISA assay, in which active RhoA (RhoA-GTP) was indexed as absorbance at 490 nm (A490). RhoA protein was used as positive control. Bars, mean ± S.D. (error bars) of duplicate measurements.

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Chemotaxis Assay, Double Knockout, MANN-WHITNEY, Migration, Activity Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: UTP- and complement C5a-induced Ca2+ transients in Gnaq/Gna11 double knockout macrophages. A, time-lapse images (90 × 90 μm) of WT macrophages loaded with the fluorescent Ca2+ indicator Cal-520. UTP was added as indicated. Scale bar, 10 μm. The intracellular Ca2+ signaling corresponding to the labeled macrophage (MΦ1) is shown below. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, time-lapse images (90 × 90 μm) of Gnaq/Gna11 dKO macrophages loaded with the fluorescent Ca2+ indicator Cal-520. UTP was added as indicated, and 22 min later, complement C5a was applied to the same cells. Scale bars, 10 μm. The intracellular Ca2+ signals corresponding to the labeled macrophages (MΦ1, MΦ2, and MΦ3) are shown below.

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Double Knockout, Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Intact complement C5a-induced Ca2+ transients in Gna12/Gna13 double knockout and Gnaq/Gna11 double knockout macrophages. A, time-lapse images (90 μm × 90 μm) of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a was added as indicated. Scale bars, 10 μm. B, intracellular Ca2+ signals corresponding to the above labeled macrophages (MΦs; A). Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. C, summary peak [Ca2+] data. n.s., not significant; Kruskal–Wallis test (n = 20–27 per group; 2–3 independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Double Knockout, Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Gna15 is redundant for complement C5a-mediated chemotaxis. A, schematic diagram highlighting that Gna15 belongs to the Gαq/Gα11 family of α-subunits. B, migration plots of WT and Gna15−/− macrophages in a chemotactic complement C5a gradient. C, 200 × 300-μm snapshot of WT and Gna15−/− macrophages in a chemotactic complement C5a gradient. D, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index), calculated by dividing the displacement along the y axis by the cumulative distance migrated. n.s., not significant; Mann–Whitney U test (n = 75 per group; three independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Chemotaxis Assay, Migration, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Complement C5a-induced Ca2+ transients are largely abolished in Gna15-deficient macrophages. A, time-lapse images (90 × 90 μm) of WT macrophages loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a and UTP were added as indicated. Scale bar, 10 μm. Below the series of four images is the intracellular Ca2+ signaling corresponding to the macrophage (MΦ) labeled MΦ1. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, time-lapse images (90 μm × 90 μm) of Gna15−/− macrophages loaded with Cal-520. Complement C5a and UTP were added as indicated. Scale bar, 10 μm. Below are traces corresponding to the labeled Gna15−/− macrophages (MΦ1 and MΦ2, respectively). C, summary peak [Ca2+] data. *, p < 0.05; Mann–Whitney U test (n = 14 for WT (two independent experiments); n = 30 for Gna15−/− (three independent experiments)).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Labeling, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Intact complement C5a-induced lamellipodial membrane spreading and Ca2+ transients in Gna15−/− macrophages. A, time-lapse images (90 × 90 μm) of WT and Gna15−/− macrophages stained with the fluorescent plasma membrane marker CellMask Orange and loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a was added as indicated. The white arrows indicate examples of lamellipodial membrane protrusion. Scale bars, 10 μm. Below is the intracellular Ca2+ signaling corresponding to the Gna15−/− macrophage labeled MΦ1. Complement C5a and UTP were added as indicated. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, box plots of projected cell area before and after application of complement C5a to WT or Gna15−/− macrophages. *, p < 0.05; Mann–Whitney U test. C, box plots of relative peak projected cell area after application of ligand-free medium (Sham) and complement C5a-containing medium to WT and Gna15−/− macrophages. *, p < 0.05; Mann–Whitney U test (n = 34 for WT and n = 29 for Gna15−/− (three independent experiments)). D, box plots of the changes in cell area (prestimulation cell area subtracted from the peak poststimulation cell area) after stimulating WT and Gna15−/− macrophages with complement C5a (n.s., not significant; Mann–Whitney U test).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Membrane, Staining, Clinical Proteomics, Marker, Labeling, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Gnb2−/− macrophages show robust complement C5a-induced cell spreading and Ca2+ transients. A, time-lapse images (90 × 90 μm) of WT and Gnb2−/− macrophages stained with the fluorescent plasma membrane marker CellMask Orange and loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a was added as indicated. The white arrows indicate examples of lamellipodial membrane protrusion. Scale bars, 10 μm. Below the series of cell morphology (CellMask Orange) images is the intracellular Ca2+ signaling corresponding to the individual macrophages labeled MΦ1. Complement C5a and UTP were added as indicated. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, summary box plots of peak cell spreading and peak intracellular [Ca2+] induced by complement C5a. n.s., not significant; Mann–Whitney U test (n = 52 per group; three independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Staining, Clinical Proteomics, Membrane, Marker, Labeling, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Gnb2−/− macrophages have decreased velocity and impaired navigation in a chemotactic complement C5a gradient. A, schematic diagram highlighting the Gβ-subunit Gβ2 (Gnb2). B, migration plots of WT and Gnb2−/− macrophages in a chemotactic complement C5a gradient. C, 200 × 300-μm snapshot of WT and Gnb2−/− macrophages in a chemotactic complement C5a gradient. D, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index), calculated by dividing the displacement along the y axis by the cumulative distance migrated. *, p < 0.05; Mann–Whitney U test (n = 75 per group; three independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Migration, Chemotaxis Assay, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Tabular summary and schematic diagram of G protein subunits involved in transducing complement C5a gradients into directed migration. A, tabular summary of results. B, schematic summary. C5aRs couple (i) directly to at least two heterotrimeric G proteins formed by Gα15 and Gαi2 subunits and possibly also Gα12/Gα13 and Gαi3 (not shown) subunits and their respective Gβγ subunits and (ii) indirectly to Gαq/Gα11-containing heterotrimeric G proteins via autocrine ATP signaling, which stimulates P2Y2Rs. The Gαi2 subunit is indispensable for chemotaxis and associates with Gβ2-containing, or possibly also Gβ1-containing, Gβγ subunits. Gαi2/Gβ2γx heterotrimeric G proteins, where x is unknown, dissociate into active (GTP-bound) Gαi2 subunits and Gβ2γx dimers following receptor activation by complement C5a. The Gβ2γx (or possibly Gβ1γx) dimers activate PI3Ks, which catalyze the conversion of PIP2 to PIP3. PIP3 is known to recruit pleckstrin homology domain–containing Rac- and Cdc42-GEFs to the membrane. Activation of Gα15-containing heterotrimeric G proteins directly by complement C5a, as well as indirect activation of Gαq/Gα11-containing heterotrimeric G proteins via autocrine ATP and UTP signaling, increases the activity of PLC-β isoforms, which catalyze the hydrolysis of PIP2 to inositol IP3 and diacylglycerol. IP3 induces Ca2+ release from the endoplasmic reticulum, but this Ca2+ signal is largely redundant for lamellipodial membrane protrusions and chemotaxis. However, we speculate that depletion of PIP2 by PLC-β isoforms and PI3Ks contributes to the formation of lamellipodial membrane protrusions by promoting the dissociation of Rac– and Cdc42-GTPase–activating proteins (GAPs). We speculate that activation of Gα12/Gα13 by complement C5a-C5aR signaling, which remains to be confirmed, increases the activity of the monomeric (small) G proteins RhoA and RhoB via RhoGEFs. Activated (GTP-bound) RhoA and RhoB promote actomyosin-dependent retraction of the trailing end of migrating cells, whereas the RhoGAP Myo9b is thought to inhibit RhoA and RhoB at the front of cells. Extracellular ATP and UTP stimulate P2Y2Rs. ATP, but not UTP, additionally activates P2X receptors (not shown), ligand-gated cation channels. ATP and UTP are rapidly degraded by surface ectonucleotidases, such as CD39, to form ligands for other purinergic receptors (not shown).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Migration, Chemotaxis Assay, Activation Assay, Membrane, Activity Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 5. FGF19 induced NET formation by facilitating complement C5a and IL-1𝛽production in iCAFs. A) Cytokine array of the media of LX-2 cells pretreated with rhFGF19 (50 ng mL−1) for 24 h. Seven factors were upregulated in the supernatant of LX-2 cells stimulated with rhFGF19. B) Quantification of NETs formed by neutrophils stimulated with complement C5a, CXCL11, IL-1𝛼, IL-1𝛽, IL-18, MIF, or PAI-1 (n = 18 RMFs from 6 experimental replicates per group). C) Intracellular and D) extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with rhFGF19 or FGF19-containing CM (n = 3 for WB, or 6 for ELISA). E) Quantification of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with FGF19-containing CM in the presence of C5a neutralizing antibody (50 ng mL−1) or IL-1𝛽neutralizing antibody (1 μg mL−1) (n = 18 RMFs from 6 experimental replicates per group). F) Representative IHC staining of C5a or IL-1𝛽expression in paired primary CRC and CRLM tissues. Scale bar: 50 μm. G) Expression of complement C5a and IL-1𝛽were higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). H) Correlation between IHC scores of FGF19 and C5a or IL-1𝛽in human CRLM tissues (n = 30). I) ChIP‒qPCR assays showed the recruitment of STAT3 to the promoter regions of C5 and IL1B (n = 3). J,K) Intracellular and L) extracellular expression of C5a and IL-1𝛽in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM and treated with or without fisogatinib (100 nм), fedratinib (10 μм), C188-9 (5 μg mL−1), and anakinra (20 mg mL−1) (n = 3 for WB, or 6 for ELISA). M) Extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected to regular medium, regular medium with rhIL-1𝛼(1 ng mL−1), FGF19-free CM, fresh FGF19-containing CM, or anakinra for 24 h (n = 6). ** or ##p < 0.01; *** or ###p < 0.001; n.s., nonsignificant. In (B), (D), (I), (L), and (M), data were subjected to Student’s t-test. In (E), data were subjected to Mann–Whitney test. In (G), data were subjected to paired Student’s t-test. See also related Figure S6, Supporting Information.

Article Snippet: Human FGF19 neutralizing antibody (AF969, R&D Systems), human IL-1β neutralizing antibody (AF-201-NA, R&D Systems), and human complement C5a neutralizing antibody (MAB2037, R&D Systems) were used in this study.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Panel A: Aliquots were separated on an 8% SDS-PAGE and then immunoblotted for the indicated proteins. Gels were cropped to focus only on the high molecular weight SDS-resistant bands. Samples also were blotted for factor B to verify cleavage as an indicator of complement activation. The 75 kDa β-chain of C3 was utilized as a loading control. Panel B: C5a ELISA of complement activation in serum. Panel C: C5a ELISA of complement activation in citrated plasma.

Article Snippet: This was followed by 4 washes and incubation with 100 ng of the detection antibody, biotinylated mouse monoclonal anti-human C5a (clone 295009, R&D Systems) for 60 minutes at room temperature with shaking.

Techniques: Clinical Proteomics, Activation Assay, SDS Page, High Molecular Weight, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: The amount of C5a generated in reconstituted C3-depleted serum samples measured by ELISA. Antibody coated sheep erythrocytes were treated with either C3-depleted serum alone or C3-depleted serum reconstituted with 1.3 mg/ml native C3, hydroxylamine treated C3 (C3-NH2OH) or C3b and incubated at 37°C for 1 hour. The supernatant was removed and C5a levels quantified by C5a ELISA. Panel B: C3-depleted serum alone or reconstituted with native C3 or hydroxylamine treated C3 (C3-NH2OH), both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG, factor B and C3. Panel C: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for C3. Panel D: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then immunoblotted C3.

Article Snippet: This was followed by 4 washes and incubation with 100 ng of the detection antibody, biotinylated mouse monoclonal anti-human C5a (clone 295009, R&D Systems) for 60 minutes at room temperature with shaking.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, SDS Page, Control, Activation Assay, Clinical Proteomics