human complement component c5a duoset elisa  (R&D Systems)

Journal: medRxiv

Article Title: Differential virulence potential of different clades of multidrug-resistant Klebsiella pneumoniae ST258

doi: 10.64898/2026.03.28.26349612

Figure Lengend Snippet: (A and B) Bacteria recovered after 30-minute exposure of 10 8 CFU/mL to 85% human serum. Limit of detection (LOD) is indicated with a dashed line. (A) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (B) 6 biological replicates per strain were tested. (C and D) C3 binding to bacteria was measured using an indirect ELISA. (C) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (D) 6 biological replicates were tested per strain, each with at least 5 technical replicates. (E) Correlation between C3 binding and serum survival of Clade 2 isolates was assessed using Spearman correlation. (F) Uronic acid was quantified from a selection of Clade 1 (n=6) and Clade 2 (n=10) isolates with 3 biological replicates per isolate. Significance was assessed with Welch’s t-test. (G) Correlation between uronic acid quantity and serum survival of 10 Clade 2 isolates was assessed using Spearman correlation. (H) OD-normalized bacteria were reacted with 5% human serum in PBS for 30 minutes. C5a from the supernatant of the reaction was quantified using a sandwich ELISA. At least two biological replicates per isolate were performed. For panels A-D and H, P ≤ 0.05: *, P ≤ 0.01: **, P ≤ 0.001: *** by Mann-Whitney test.

Article Snippet: C5a quantification was performed using the Human Complement Component C5a DuoSet ELISA from R&D systems.

Techniques: Bacteria, Binding Assay, Indirect ELISA, Selection, Sandwich ELISA, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Prognostic potential of C5a as a clinical marker. ( a ) Schematic illustration depicting the informatics analysis conducted on primary glioblastoma (GBM) samples obtained from the Severance cohort. ( b ) Volcano plot showing differentially expressed genes (DEGs) in the C5a High group compared to those in the C5a Low group. Upregulated, downregulated, and insignificantly altered DEGs are indicated. Genes highlighted in white boxes are significantly associated with the tumor microenvironment (TME) and inflammation. ( c ) Heatmap illustrating the expression profiles of statistically significant DEGs (P-adj < 0.05) between the C5a High and C5a Low groups. ( d ) Gene Ontology enrichment analysis for biological processes related to DEGs in the C5a High group versus the C5a Low group. ( e ) Gene set enrichment analysis (GSEA) plot showing the enrichment of hallmark TME and inflammation-related gene sets in the C5a High group relative to the C5a Low group. ( f ) Kaplan–Meier survival curve comparing overall survival between patients with GBM in the C5a High group (n = 61) and those in the C5a Low group (n = 35). Patients in the C5a High group exhibited significantly worse survival outcomes compared to those in the C5a Low group (P = 0.027).

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Marker, Expressing

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Evaluation of cell viability, ATP levels, and molecular changes in glioblastoma tumorspheres treated with C5a-enriched conditioned media and W54011 . ( a , b ) Assessment of cell viability and ATP levels in three glioblastoma (GBM) tumorspheres (TS15-88, TS14-15, and U87) treated with C5a-enriched conditioned media (CM) alone or in combination with W54011 at varying concentrations (0–30 μM). Graphs show the dose–response curve used to calculate IC 50 values (solid bars, control [Cont]; bars with slashed lines, C5a-enriched CM-treated). The “0 μM” W54011 condition refers to treatment with CM in the absence of W54011 (i.e., CM + 0 W54011 ), which served as a positive control to evaluate the full effect of C5a without receptor inhibition. ( c ) Quantification of C5a levels in CM derived from GBM tumorspheres using ELISA. The cells were treated with C5a-enriched CM alone or in combination with W54011 at concentrations ranging from 0–7.5 μM (solid bar, Cont; bar with slashed lines, C5a-enriched CM-treated). ( d ) Western blotting analysis of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at indicated concentrations (0, 2, 5, and 7.5 μM). Blots were probed with antibodies against pro-caspase-3, Bcl-2, Bax, pro-PARP, cleaved PARP, and GAPDH. ( e ) Heatmap illustrating changes in the expression of genes within the GBM amplification marker gene set between GBM tumorspheres treated with C5a-enriched CM alone or with 7.5 μM W54011 . Statistical analysis was performed using one-way analysis of variance, followed by Tukey’s post hoc test. Data are presented as means ± standard deviation, with statistical significance indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) to denote differences between groups or relative to control conditions; NS, not significant.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Control, Positive Control, Inhibition, Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Amplification, Marker, Standard Deviation

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Reduction in stemness and morphological changes in glioblastoma tumorspheres following W54011 treatment in the presence of C5a-enriched conditioned media. ( a ) High-throughput brightfield micrographs of the three glioblastoma (GBM) tumorspheres (TS15-88, TS14-15, and U87) treated with C5a-enriched conditioned media (CM) alone or in combination with W54011 at various concentrations (0–7.5 μM), starting from an initial seeding density of 200 cells. The 0 μM W54011 group represents CM-treated cells without antagonist exposure. ( b ) Frequency of stem-like cells within populations of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at various concentrations (0–7.5 μM), as measured using the extreme limiting dilution assay at 14 days post-seeding. ( c ) Western blotting analysis of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at concentrations of 0, 2, 5, and 7.5 μM. Blots were probed with antibodies against Nestin, Sox2, PDPN, OCT3/4, and GAPDH. ( d ) Heatmap showing changes in gene expression within the GBM stemness marker gene set in GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at 7.5 μM. ( e ) Representative confocal micrographs demonstrating colocalization of GFAP (red) and C5a (green) in GBM tumorspheres treated with C5a-enriched CM alone or in combination with 7.5 μM W54011 . Scale bars = 50 μm. Cont, control.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: High Throughput Screening Assay, Limiting Dilution Assay, Western Blot, Gene Expression, Marker, Control

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Inhibition of invasion by W54011 in glioblastoma tumorspheres exposed to C5a-enriched conditioned media. ( a ) Time-lapse images of spheroid invasion in three glioblastoma (GBM) tumorspheres (TS15-88, TS14-15, and U87) within a matrix gel. Representative images show spheroids at baseline (0 h, left corner) and after 72 h of treatment with C5a-enriched conditioned media (CM) alone or in combination with W54011 at various concentrations (0–7.5 μM). “0 μM W54011 ” indicates treatment with C5a-enriched CM alone. Scale bars = 200 µm. ( b ) Quantification of the invasion area of individual spheroids (solid bar, control [Cont]; bar with slashed lines, C5a-enriched CM-treated). ( c ) Western blotting analysis of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at concentrations of 0, 2, 5, and 7.5 μM. Cell lysates were probed with antibodies against Zeb1, N-cadherin, β-catenin, CD133, CD44, Snail, Twist, and GAPDH. ( d ) Heatmap depicting gene expression changes in the GBM invasion marker gene set in GBM tumorspheres treated with C5a-enriched CM alone or with 7.5 μM W54011 . Statistical analysis was performed using one-way analysis of variance, followed by Tukey’s post hoc test. Results are presented as means ± standard deviation, with significance indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), highlighting differences between groups or compared to control conditions.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Inhibition, Control, Western Blot, Gene Expression, Marker, Standard Deviation

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Effect of W54011 on tumor growth in an in vivo model with C5a-enriched conditioned media. ( a ) Schematic illustrating the xenograft model design and treatment protocol. “Adaptation” denotes a one-week acclimation period following animal arrival. “Bolt” indicates surgical implantation of a cranial guide-screw at week –1, enabling orthotopic cell injection at week 0. Eight-week-old BALB/c nude mice were divided into four groups: TS (n = 8), TS + W54011 (n = 5), TS + CM (n = 8), and TS + CM + W54011 (n = 10). Orthotopic xenografts were established with TS15-88-luciferase cells (5 × 10 5 ) alone or with TS15-88 (2.5 × 10 5 ) and tMSLC0903-01 (2.5 × 10 5 ). Mice were treated with or without W54011 before surgical orthotopic implantation to assess the effects of C5a-enriched conditioned media (CM) and W54011 in vivo. ( b ) Tumor growth was monitored using an IVIS Lumina II in vivo imaging system starting on day 8 and every week thereafter. Bioluminescence images show tumor signals obtained at 13 weeks. ( c ) Representative magnetic resonance imaging (MRI) scans at 18 weeks post-inoculation. MRI was performed once at the experimental endpoint to confirm tumor presence and assess volume. Red lines indicate tumor volumes. ( d ) Bar graph quantifying MRI tumor volumes (open circle, DMSO-treated; open triangle, W54011 -treated; white box, only TS15-88 injected; blue box, TS15-88 and tMSLC09030-1 co-injected). ( e ) Staining with 3,3′-diaminobenzidine was used to assess the expression of C5a, N-cadherin, and vimentin in tumor tissues from the TS, TS + CM, and TS + CM + W54011 groups. The scale bars represent 200 µm (top) and 50 µm (bottom), respectively. ( f ) Kaplan–Meier survival analysis of the mouse models. The P-value measured when comparing TS + CM (n = 5) to TS + W54011 (n = 8) was 0.00053, and that when comparing TS + CM (n = 5) to TS + CM + W54011 (n = 10) was 0.032. Statistical analysis was conducted using one-way analysis of variance, followed by Tukey’s post hoc test. Results are presented as means ± standard deviation, with significance indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), highlighting differences between groups or compared to control conditions.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: In Vivo, Injection, Luciferase, In Vivo Imaging, Magnetic Resonance Imaging, Staining, Expressing, Standard Deviation, Control

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Schematic illustration of W54011 -mediated restoration of the C5a-altered glioblastoma tumor microenvironment. Tumor mesenchymal stem-like cells (tMSLCs) secrete C5a, which activates C5aR1 signaling in glioblastoma cells, driving enhanced proliferation, stemness, and invasiveness. This C5a-enriched microenvironment promotes tumor progression and malignant phenotypes. Pharmacologic inhibition of C5aR1 with W54011 suppresses C5a-induced downstream signaling and restores tumor cells toward a less aggressive state.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Inhibition

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) Shown are representative fields from 3 independent experiments. Complement proteins C3a and C5a increased the expression of ECM proteins in THP1 macrophages, BMDMs, and HK2 proximal tubule cells after 72 hours of treatment in serum-free medium. Scale bars: 50 μm. ( B – D ) The scatter plots show the mean staining intensity per THP-1 macrophage ( B ), BMDM ( C ), and HK2 proximal tubule cell ( D ), normalized to expression levels in their respective vehicle-treated groups. Each data point corresponds to quantified fluorescence intensity in a single field of view (FOV) from the microscope, and the larger dots represent the average of FOVs in biological replicates, each of which is color coded. RT-qPCR analysis of ECM protein coding genes were measured in BMDMs ( E ) and in HK2 proximal tubule cells ( F ). Gene expression was normalized to the expression of 18S ribosomal RNA in the same sample and then normalized to the expression level of vehicle-treated group ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: Circulating immune complexes and Abs planted directly within glomerular and tubulo-interstitial regions of the kidneys may fix complement, resulting in complement activation. The alternative pathway may further amplify complement activation within the kidneys. The products of C3 and C5 convertases, including the anaphylatoxins C3a and C5a, engage cognate receptors on a wide spectrum of immune cells, leading to immune cell activation, release of cytokines and chemokines, and acute inflammation, leading to high AI, as depicted on the left. Long-standing, unresolved complement activation and eventual formation of MAC may additionally engage and activate more immune and renal-resident cells, leading to tissue damage and repair, ECM deposition, and renal fibrosis, leading to high CI, as depicted on the right.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: P-Rex1 controls phagocytosis and the killing of bacteria by murine neutrophils independently of its catalytic activity

doi: 10.3389/fimmu.2025.1591006

Figure Lengend Snippet: P-Rex1 mediates the killing of S. aureus by neutrophils independently of its catalytic Rac-GEF activity, whereas chemotaxis, ROS, and NETs require its Rac-GEF activity. (A) Bactericidal activity. Purified neutrophils from Prex1 –/– (red squares), Prex1 GD (green triangles), and wild type mice (grey circles) were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before incubation with serum-opsonized S. aureus for 90 min at a ratio of 1.5 bacteria per neutrophil. Heat-killed neutrophils were used as negative controls. Surviving bacteria were grown overnight and CFU enumerated. The % killing of bacteria by live neutrophils compared to heat-killed controls is plotted. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are one-way ANOVA with Tukey’s multiple comparisons test on log-transformed raw data; black p-values are significant, grey p-values non-significant. (B) Chemotaxis. Bone marrow cells from Prex1 –/– , Prex1 GD , and wild type mice were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before being stimulated with 3 nM C5a in transwell filters for 40 min, or mock stimulated. Transmigrated cells were analyzed by flow cytometry in parallel to control cells, using Ly6G hi /Mac1 hi staining to identify neutrophils. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons test on raw data. (C) fMLP-stimulated ROS production. Purified neutrophils as in (A) were primed with 1 μg/ml LPS for 90 min and then stimulated with 3 µM fMLP (filled symbols), or mock-stimulated (open symbols). ROS production was measured by real-time chemiluminescence assay with luminol and HRP for extra- and intracellular ROS. Left-hand panel shows luminometer traces from one representative experiment; right-hand panel shows the quantification as AUC over 2 min. Data are mean ± SEM of 3–5 independent experiments; each symbol represents the mean AUC from one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on log-transformed raw data. (D) S. aureus -stimulated intracellular ROS. Neutrophils were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min in the presence of 50 units/ml SOD and 2000 units/ml catalase to scavenge extracellular ROS and were then stimulated with S. aureus at a ratio of 10 bacteria per neutrophil (filled symbols), or mock-stimulated (open symbols). ROS production was measured as in (C) except without HRP and in the presence of SOD and catalase, and quantification was done over 60 min. Data are mean ± SEM of 4 independent experiments; statistics are two-way ANOVA with Sidak’s multiple comparisons tests. (E) Formation of NETs. Neutrophils were seeded onto glass slides and allowed to adhere for 30 min before stimulation with serum-opsonised S. aureus at a ratio of 10 bacteria per neutrophil (closed symbols), or mock stimulation (open symbols). Non-cell permeable Sytox Green and cell-permeable Hoechst 33342 DNA dyes were added to samples 15 min before the end of the incubation, and cells were live-imaged by wide-field microscopy. Left-hand panel shows representative images from one experiment after 120 min stimulation or mock stimulation. Insets are magnifications of the indicated areas. Red arrows highlight NETs, white arrows dead cells without NETs. Right-hand panel shows quantification of NETs by ImageJ. Data are mean ± SEM of 3–4 independent experiments. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on raw data; significant p-values between and Prex1 –/– and wild type are indicated in red, and between Prex1 GD and wild type in green. For all panels, closed symbols show stimulated cells, open symbols mock-treated cells.

Article Snippet: Cells were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min at 37°C, pipetted into transwell filters (400 μl/filter) in a 24-well plate containing HBSS ++++ (600 μl/well) in the presence or absence of 3 nM complement factor C5a anaphylatoxin (C5a, R&D Systems, 2037-C5-025), and incubated for 40 min at 37°C.

Techniques: Activity Assay, Chemotaxis Assay, Purification, Incubation, Bacteria, Transformation Assay, Flow Cytometry, Control, Staining, Chemiluminescence Immunoassay, Microscopy