Journal: Biochemistry and Biophysics Reports

Article Title: Complement dependent TNFα production in neutrophil-like HL60 cells

doi: 10.1016/j.bbrep.2023.101465

Figure Lengend Snippet: Zymosan-treated human serum-induced TNFα production in neutrophil-like HL60 cells. HL60 cells were induced to differentiate into neutrophil-like cells by treatment with 2.5 μM ATRA for 0–5 days. (A) Flow cytometric analysis of the cell surface expression of CR3, C3aR, or C5aR1 in day 0, 1, 3, and 5 ATRA-treated HL60 cells. (B) Day 0–5 ATRA-treated HL60 cells were either untreated (NT) or incubated with 10% human serum or zymosan-treated human serum for 4 h, and the whole cell lysates (Cell) and culture supernatants (Sup.) were then collected. The protein samples were separated in SDS-PAGE, and TNFα and β-actin were detected by immunoblot analysis. RPMI, Human serum, and ZHS in diagonal letters indicate samples of RPMI-1640 medium, RPMI-1640 containing 10% zymosan-treated human serum, or human serum mixed with 1 × SDS-PAGE sample loading buffer, respectively. (C, D) Day 5 ATRA-treated HL60 cells were either untreated (NT) or incubated with 10% human serum or zymosan-treated human serum for indicated time points, and the whole cell lysates (Cell) and culture supernatants (Sup.) were then collected. (E) Day 5 ATRA-treated HL60 cells and PMNs were either untreated (NT) or incubated with 10% human serum (HS) or zymosan-treated human serum (ZHS) for 4 h, and the whole cell lysates (Cell) and culture supernatants (Sup.) were then collected. The protein samples were separated in SDS-PAGE, and TNFα and β-actin were detected by immunoblot analysis. The blot is a representative of 3 independent experiments.

Article Snippet: Anti-C3a Receptor (hC3aRZ8) FITC monoclonal antibody (#130-108-108) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Incubation, SDS Page, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Complement dependent TNFα production in neutrophil-like HL60 cells

doi: 10.1016/j.bbrep.2023.101465

Figure Lengend Snippet: Schematic diagram of the hypothesis that complement-induced TNFα production in neutrophil-like HL60 cells. The complement activation product C3a binds to C3aR and induces TNFα production in neutrophil-like HL60 cells. The TNFα production is enhanced by intracellular Ca 2+ elevation and is negatively regulated by fibrinogen or fibronectin coating.

Article Snippet: Anti-C3a Receptor (hC3aRZ8) FITC monoclonal antibody (#130-108-108) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Activation Assay

Journal: The FASEB Journal

Article Title: IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

doi: 10.1096/fj.201700289R

Figure Lengend Snippet: IL-17A activates C′ by modulating the mRNA and protein expression of the C′ components in vitro. A) Normal human primary AECs were exposed to recombinant human IL-17A (100 ng/ml; 24 h). Subsequent to RNA isolation, cDNA was generated and subjected to real-time quantitative PCR analyses. Means ± sem; Student’s t test. B) Protein lysates from AECs exposed to recombinant human IL-17A (100 ng/ml; 4 h) were subjected to immunoblot analysis against C3aR, C5aR, DAF, and vinculin. C) Conditioned medium collected from AECs exposed to the indicated doses of IL-17A for 4 h was analyzed for active C3a by ELISA. Means ± sem. D) AECs exposed to IL-17A (100 ng/ml; 4 h) were methanol fixed, immunostained for DAF, and counterstained with DAPI. Results represent 3 independent experiments. Scale bars, 100 µm.

Article Snippet: The antibodies used for immunoblot analysis or immunofluorescent labeling are as follows: decay-accelerating factor (DAF)-clone H-319 (sc-9156); and vinculin-clone7F9 (sc-73614) (Santa Cruz Biotechnology), C3a receptor (C3aR; NBP1-61567), and C5a receptor (C5aR; NBP2-15649; Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, In Vitro, Recombinant, Isolation, Generated, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

doi: 10.1096/fj.201700289R

Figure Lengend Snippet: IL-17A deficiency protects against BLEO-induced local C′ activation. The BALF samples collected for the analysis shown in Fig. 2 were also analyzed for C3a (A), C5a (B), and C5b-9 (C) levels by ELISA (n = 7–8/group). Means ± sem. One-way ANOVA, Newman-Keuls (A, C), and Bonferroni (B).

Article Snippet: The antibodies used for immunoblot analysis or immunofluorescent labeling are as follows: decay-accelerating factor (DAF)-clone H-319 (sc-9156); and vinculin-clone7F9 (sc-73614) (Santa Cruz Biotechnology), C3a receptor (C3aR; NBP1-61567), and C5a receptor (C5aR; NBP2-15649; Novus Biologicals, Littleton, CO, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: ( A ) Schematic diagram showing the complement classical pathway function in synapse removal and neuroinflammation via C3b and C3a (created with BioRender.com ). C1 complex (C1q, C1r, and C1s) activation and binding to a synapse leads to deposition of C4b and C2b fragments (C3 convertase), resulting in C3b deposition which is recognized by CR3 on microglia, and release of soluble C3a, recognized by C3aR on microglia. ( B ) Study design showing mouse genotypes and timing of experimental measurements: MRI, immunohistochemistry, and behavior. ( C ) Representative MRI images in 6-month-old (top) and 9-month-old (bottom) male mice with genotypes as indicated. Red arrows in 9-month-old TauP301S mouse images indicate enlargement of ventricles, and white arrows indicate hippocampal atrophy. ( D) Longitudinal MRI quantification of volume changes of ventricles, neocortex, and hippocampus at 6 and 9 months compared with 3 months of age in mice with indicated genotypes. ( E ) Longitudinal MRI quantification of ventricle, neocortex, and hippocampus volumes after first normalizing to total brain volume, showing changes of normalized volumes at 6 and 9 months compared with 3 months mice with indicated genotypes. n = 9-12 male mice per genotype. Data are represented by mean +/- SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: Activation Assay, Binding Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: Single cell RNA-sequencing was performed on brain tissues from wild-type (C3aR +/+) and C3aR knockout (C3aR -/-) mice treated with lipopolysaccharide (LPS) or vehicle (control). (A) UMAP plot displaying the clustering of single cells across both genotypes and treatment conditions, color-coded by the major cell types. (B) UMAP plot overlaid with C3ar1 expression (log-normalized counts), demonstrating that C3ar1 expression is restricted to microglia and PVMs. (C) UMAP plot highlighting the C3aR +/+ and C3aR -/- cells derived from animals treated with LPS. (D) Bar plot quantifying the number of differentially expressed genes (DEGs) across all conditions, separated by cell type. (E) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR WT microglia. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (F) Four-way comparison of DE genes in LPS treated microglia in C3aR +/+ (x-axis) vs. C3aR -/- genotype (y-axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis. (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x. (G) Volcano plot showing the differential gene expression between C3aR WT and KO microglia in LPS treated mice. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in C3aR KO and blue for downregulated in C3aR KO. (H) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR KO PVM. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (I) Four-way comparison of DE genes in LPS treated microglia in C3aR +/+ (x-axis) vs. C3aR -/- genotype (y-axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis. (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x. (J) Volcano plot showing the differential gene expression between C3aR WT and KO PVM in LPS treated mice. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in C3aR KO and blue for downregulated in C3aR KO.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Knock-Out, Control, Expressing, Derivative Assay, Gene Expression, Comparison, Quantitative Proteomics

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: (A) UMAP plot displaying the sub-clustering of microglia into transcriptionally distinct subpopulations, identified based on single cell RNA-seq analysis. (B) Average expression of genes representing specific microglial states calculated across the dataset and represented in the form of a feature plot. (C) Proportion of each microglial subpopulation across treatment and genotype groups expressed as a percentage of total microglia, illustrating a shift toward activated microglia in response to LPS treatment, independent of C3aR genotype. Differential abundance statistics * FDR < 0.05; ** FDR <0.01 and *** FDR< 0.001. (D) Dotplot of the top marker genes of each microglial subpopulation highlighting the transcriptional states associated with activated and proliferative states. (E) Bubble plot representing the results of a Gene Ontology (GO) term enrichment analysis using EnrichGO. X-axis denotes microglial subclusters and y-axis denotes enriched GO terms. The size of the bubble reflects the number of genes contributing to enrichment and the color represents the adjusted p-value.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Expressing, Marker

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: (A) UMAP plot displaying the sub-clustering of astrocytes into transcriptionally distinct subpopulations, identified based on single cell RNA-seq analysis. (B) Dotplot of the top marker genes of each astrocyte subpopulation highlighting the transcriptional states associated with LPS treatment. (C) Bubble plot representing the results of a Gene Ontology (GO) term enrichment analysis using EnrichGO. X-axis denotes astrocyte subclusters and y-axis denotes enriched GO terms. The size of the bubble reflects the number of genes contributing to enrichment and the color represents the adjusted p-value. (D) Proportion of each astrocyte subpopulation across treatment and genotype groups expressed as a percentage of total astrocytes, illustrating a shift toward an inflammatory state in response to LPS treatment, independent of C3aR genotype. Differential abundance statistics * FDR < 0.05; ** FDR <0.01 and *** FDR< 0.001. (E) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR WT astrocytes. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (F) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR KO astrocytes. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (G) Four-way comparison of DE genes in LPS treated astrocytes in C3aR +/+ (x-axis) vs. C3aR -/-genotype (y- axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Marker, Gene Expression, Comparison, Quantitative Proteomics

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: (A) UMAP plot displaying the sub-clustering of oligodendrocyte lineage cells into transcriptionally distinct subpopulations, identified based on single cell RNA-seq analysis. (B) Proportion of each oligodendrocyte lineage subpopulation across treatment and genotype groups expressed as a percentage of total oligodendrocyte lineage cells, illustrating a shift toward an inflammatory state in response to LPS treatment, independent of C3aR genotype. Only cell populations with proportions that are greater than 1% of the total OL lineage cells are shown although all cells were included in the calculation. Differential abundance statistics * FDR < 0.05; ** FDR <0.01 and *** FDR< 0.001. (C) Dotplot of the top marker genes of each astrocyte subpopulation highlighting the transcriptional states associated with LPS treatment. (D) Bubble plot representing the results of a Gene Ontology (GO) term enrichment analysis using EnrichGO. X-axis denotes astrocyte subclusters and y-axis denotes enriched GO terms. The size of the bubble reflects the number of genes contributing to enrichment and the color represents the adjusted p-value. (E) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3arR WT oligodendrocytes. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment (F) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR KO oligodendrocytes. (G) Four-way comparison of DE genes in LPS treated oligodendrocytes in C3aR +/+ (x-axis) vs. C3aR -/- genotype (y-axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Marker, Gene Expression, Comparison, Quantitative Proteomics

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3aR ameliorates rotenone-induced neurodegeneration, synaptic loss, α-synuclein phosphorylation and cognitive deficits in mice. C57BL/6 mice were treated with rotenone for 3 weeks to establish PD models, with 30 min of SB290157 (C3aR inhibitor, 1 mg/kg, i.p.) administration after each rotenone injection. (A) Concentrations of C3a in the hippocampus of Con and Rot mice, measured by enzyme-linked immunosorbent assay (ELISA). n = 6. (B) Representative Western blot images and densitometric quantification of C3aR protein levels in the hippocampus and rotenone mice. n = 4. (C) The mRNA levels of C3aR in vehicle and rotenone mice. (D) Representative images of double-immunofluorescence staining with C3aR and Iba-1 or Neu-N antibodies and (E, F) the quantification of C3aR + Iba-1 + and C3aR + Neu-N + cells in vehicle and rotenone mice. n = 3. (G) The quantification of Neu-N + cell number and (H) optical density of PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. (I) The representative images of Neu-N and PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. n = 6. (J, K) Representative bands of Neu-N, PSD95 and ser129-phosphorylated α-synuclein and the quantification of blots in rotenone mice with or without C3aR inhibitor treatment. n = 4. (L) Representative images of ser129-phosphorylated α-synuclein and (M) the quantification of staining density in rotenone mice with or without C3aR inhibitor treatment. n = 3. (N–Q) Escape latency, traveled distance, first platform crossing latency and platform crossing number in rotenone mice with or without C3aR inhibitor treatment. n = 15; ∗ p < 0.05, ∗∗ p < 0.01 for comparison between Rot & Rot + C3aR inhibitor groups; Scale bar = 50 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Phospho-proteomics, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Double Immunofluorescence Staining, Staining, Comparison

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis suppresses microglia-mediated neuroinflammation in rotenone-treated mice. WT mice, C3 −/− mice, and C3aR inhibitor (SB290157)-treated mice were exposed to rotenone for 3 weeks to induce PD-related neuroinflammation. (A) Representative images of Iba-1 immunostaining and (B) the quantification of staining density in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 6. (C) The mRNA levels of iNOS, IL-1β and TNFα in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. (D) Representative bands of p-MAPK, MAPK, p-p65, p65, p-IκBα and IκBα and the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4. ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Immunostaining, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis mitigates microglia-mediated abnormal synaptic pruning in rotenone-treated mice. WT mice, C3 −/− mice, and SB290157-treated mice were subjected to rotenone for 3 weeks. (A) Representative images of double fluorescence staining with anti-Iba-1 and anti-CD68 antibodies and (B) the quantification of staining in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 6. (C) Representative bands of PSD95, p-CREB, CREB, mBDNF and TrkB and (D) the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4. ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Fluorescence, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis attenuates rotenone-induced activation of dark microglia and related secretion of toxic lipids in vivo and in vitro. (A) Representative bands of p-eIF2α, eIF2α, PKR, PERK and ATF4 and (B) the quantification of blots in mice. n = 4. (C) Representative images of double-immunofluorescence staining with p-eIF2α and Iba-1 antibodies in mice. n = 3. (D) BV2 microglial cells were treated with C3 protein (with or without SB290157 or NOX2 inhibitor GSK2795039) for 24 h. Representative bands of p-eIF2α, eIF2α, PKR, PERK and ATF4 and the quantification of blots in BV2 cells were shown. (E) The mRNA levels of atf4 and asns in BV2 cells. n = 3. (F) 3D principal component analysis (PCA) score plot showing separation of lipid profiles. (G) The heatmap shows the differential lipid metabolites among groups. (H) Venn diagram showing the overlap of differential lipid metabolites. (I) Classification of differential lipid metabolites at the major category level between C3_C3aR vs. C3 and C3 vs. Control groups. (J) Subclass distribution of differential lipid metabolites between C3_C3aR vs. C3 and C3 vs. Control groups. n = 5. SP: sphingolipids; GL: glycerolipids; FA: fatty acyls; DL: derivatized lipids (biotinylation, diazomethane). ST: sterol lipids. PR: prenol lipids. GP: glycerophospholipids. ∗∗ p < 0.01; Scale bar = 50 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activation Assay, In Vivo, In Vitro, Double Immunofluorescence Staining, Control

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Blocking C3/C3aR axis suppresses rotenone-induced PANoptosis in mice. (A) Representative images of TUNEL staining and (B) the quantification of TUNEL + cells in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. (C) Representative bands of Bcl-2, Bax, active-caspase-3, p-MLKL, MLKL, p-RIP1, RIP1, p-RIP3, RIP3, NLRP3, caspase-1 and GSDMD and (D) the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4; ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Blocking Assay, TUNEL Assay, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: C3–C3aR axis promotes PANoptosome formation and PANoptosis through z-DNA-ZBP-1 interaction via mitochondrial ROS. SH-SY5Y neuronal cells were treated with different concentrations of C3 protein (with or without SB290157, apoptosis inhibitor Z-VAD, necroptosis inhibitor Nec-1, inflammasome inhibitor MCC950, ROS scavenger NAC, NADPH oxidase inhibitor apocynin, mtROS scavenger mito-TEMPO, z-DNA inhibitor CQ, or z-DNA promoter CeCl 3 ) for 24 h. (A) The survival rate of cells treated with different concentrations of C3 protein. (B) The survival rate of cells treated with C3 with or without different inhibitors. (C) Representative pictures of ZBP-1, AIM2, RIP3 and caspase-1 interaction using COIP. (D) The ratio of A260/A295 absorbance. (E) Representative images of DHE staining and quantification of DHE density in SH-SY5Y cell among groups. (F) Representative images of z-DNA fluorescence staining and quantification of density in SH-SY5Y cell among groups. (G) Representative blots of z-DNA and ZBP-1 and quantification of blot density in SH-SY5Y cell among groups. (H) Representative images of double-immunofluorescence staining using antibodies against z-DNA and ZBP-1 and quantification of ration of z-DNA + ZBP-1 + /ZBP-1 + cells in SH-SY5Y cell among groups. (I) Representative blots of z-DNA and ZBP-1 interaction using COIP. (K) The survival rate of cells treated with C3 with or without apocynin, NAC, mito-TEMPO, CQ and CeCL 3 . n = 3; ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm in (E) and 25 μm in (F) and (H).

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Fluorescence, Double Immunofluorescence Staining

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A, B) C3 (A) and C3aR1 (B) mRNA levels in the parahippocampal gyrus of NCI (n=32), MCI (n=34), or AD (Dementia, n=160). Spearman’s correlation, ρ (C3)=0.36, p<2.6E-08; ρ (C3aR)=0.33, p<3.4E-07.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A)Representative GFAP and Iba1 immunostainings with DAPI in the hippocampus of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. Scale bar: 50 μM.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Representative Western blots of PHF1- and CP13-positive phospho-tau and total tau in PS19 and PS19/C3aR KO mouse brains at 9 months. γ-tubulin is used as a loading control.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Western Blot

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Representative synaptophysin (Syp) and Syp/NeuN co-immunostaining in area CA3 of hippocampus of WT, C3aR KO, PS19, and PS19/C3aR KO mice at 9 months. Scale bar: 50 μM.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Immunostaining

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Unsupervised clustering gene expression heatmap from RNAseq analysis of hippocampal tissues of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. 1726 DEGs were identified by comparing the PS19 vs WT group (1155 genes upregulated, red, and 571 downregulated, blue). n=4-5/genotype, adjusted p<0.01.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Expressing

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Gene-set enrichment analyses (GSEA) of the microglia sensome gene list in PS19 vs WT (left, NES=2.81, FDR<0.001) and in PS19/C3aR KO vs PS19 (right, NES= −3.0, FDR<0.001) comparisons. NES: normalized enrichment score; FDR: false discovery rate.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A, B) Total STAT3 (A) and Y705-phospho-STAT3 (B) levels in 9 month-old WT (n=10), C3aR KO (KO, n=10), PS19 (PS, n=7), and PS19/C3aR KO (PS/KO, n=7) mice measured by RPPA (mean ± s.e.m). The asterisks above PS19 are for PS19 vs WT comparison; those above PS19/C3aR KO are for PS19/C3aR KO vs PS19 comparison. Student｀s t-test. *p<0.05; **p<0.01.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Scientific Reports

Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

doi: 10.1038/s41598-018-28143-0

Figure Lengend Snippet: C3a causes abnormal deposition of ECM by hfRPE cells, which is prevented by C3aR antagonist. TEM images show transverse sections of hfRPE cultured on transwells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. ( d ), ( e ), and ( f ) are higher magnifications from white squares in ( a ), ( b ), and ( c ) respectively. C3a addition results in the accumulation of collagen fibers (white arrowheads) and wide-spaced collagen (black arrowheads) underneath the RPE cells. The addition of C3aR antagonist prevents the accumulation of sub-RPE deposits caused by C3a ( g – i ). Scale bars a–c: 2 µm, d–i: 500 nm.

Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

Techniques: Cell Culture

Journal: Scientific Reports

Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

doi: 10.1038/s41598-018-28143-0

Figure Lengend Snippet: C3aR antagonist prevents formation of basal deposits caused by C3a. SEM images of decellularized transwells show exposed basal deposits made by hfRPE cells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. C3a triggers the accumulation of deposits made of ECM fibers underneath the RPE. The abnormal deposition is prevented by the addition of C3aR. Scale bars a–c: 100 µm, d–i: 10 µm.

Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

Techniques:

Journal: Scientific Reports

Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

doi: 10.1038/s41598-018-28143-0

Figure Lengend Snippet: C3a induces overexpression of C3aR in the basolateral membrane of the RPE. ( a ) Immunolabeling with C3aR antibodies of hfRPE cells treated with different doses of C3a (C3aR-ant) or C3a + C3aR antagonist (C3aR + ant) for 2 weeks. Z-stack was built from images taken every 0.5 µm with confocal microscope. 90° projections show that C3aR is expressed on the basal-lateral membrane of RPE. Scale bars 50 µm. ( b ) Average quantification of C3aR fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a + C3aR antagonist (C3aR + ant). (ANOVA, n = 4/C3aR-ant and n = 4/C3aR + ant treatment. Data represented as mean ± SEM. *p < 0.05, **p < 0.01). ( c ) mRNA levels of C3aR of hfRPE cells treated with different doses of C3a for 24 hours (ANOVA, n = 6. Data presented as mean ± SD. **p < 0.01).

Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

Techniques: Over Expression, Immunolabeling, Microscopy

Journal: Scientific Reports

Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

doi: 10.1038/s41598-018-28143-0

Figure Lengend Snippet: C3a causes specific deposition of Col IV and Col VI underneath the RPE that is prevented by C3aR antagonist. Immunolabeling with antibodies for ( a ) Col IV, ( c ) Col VI, ( e ) Col I, and ( g ) EFEMP1 of hfRPE cells treated with different doses of C3a or C3a + C3aR antagonist for 2 weeks. Images were taken with confocal. Orthogonal views show the basal deposition of the ECM proteins (left: apical, right: basal). Scale bars 50 µm. Average quantification of ( b ) Col IV, ( d ) Col VI, ( f ) Col I, and ( h ) EFEMP1 fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a plus C3aR antagonist (C3aR + ant) for 2 weeks. ( i ) Average quantification of immunostainings for ECM proteins after 4 weeks of treatment with C3a. (ANOVA. Data represented as mean ± SEM. n = 6/C3a dose *p < 0.05, **p < 0.01). ( j ) mRNA expression of COL4 and COL6 normalized to GAPDH after treatment with C3a for 2 weeks in the absence and presence (pattern bar) of C3aR antagonist (ANOVA. n = 3/−C3aR ant and n = 3/+ C3aR ant. Data represented as mean ± SD, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

Techniques: Immunolabeling, Expressing

Journal: Scientific Reports

Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

doi: 10.1038/s41598-018-28143-0

Figure Lengend Snippet: C3a does not make changes in calcium mobilization within the RPE cytosol. ( a ) Confocal fluorescent images of hfRPE cultures treated with different doses of rhC3a only (−C3aR ant) or rhC3a plus C3aR antagonist (+C3aR ant) after incubation with Fluoforte dye®. Scale bars 50 µm. ( b ) Quantification of calcium influx in hfRPE cultures treated with different doses of rhC3a (black bars) or rhC3a plus C3aR antagonist (grey bars), using fluorescent microscope or ( c ) microplate reader. (ANOVA, n = 8/treatment. Data represented as mean ± SD).

Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

Techniques: Incubation, Microscopy

Journal: Scientific Reports

Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

doi: 10.1038/s41598-018-28143-0

Figure Lengend Snippet: C3a decreases proteasome activity in the RPE, which is restored by the addition of C3aR antagonist. ( a ) Confocal images of UPP overall activity in hfRPE cells stimulated with different doses of C3a with or without C3aR antagonist for 72 hours stained with the probe MV-151. Control epox. = control samples treated overnight with epoxomicin. Scale bars 25 µm. Quantification of UPP overall activity after stimulation with different doses of C3a for ( b ) 2 weeks or ( c ) 72 hours with the probes MV-151 and LWA300, respectively. ( d ) mRNA expression of UPP subunits of hfRPE cells treated for 24 h with different doses of C3a normalized to GAPDH . (2-way ANOVA, n = 3/dose. Data represented as mean ± SD. *p < 0.05, **p < 0.01). White bars: control (C3a 0 ng/ml), grey bars (C3a 50 ng/ml), black bars (C3a 100 ng/ml).

Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

Techniques: Activity Assay, Staining, Expressing

Journal: Scientific Reports

Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

doi: 10.1038/s41598-018-28143-0

Figure Lengend Snippet: C3a treatment results in increased MMP-2 activity by RPE cells. ( a ) mRNA expression of MMP-2 and TIMP-3 measured by qRT-PCR and normalized to GAPDH in cultures of hfRPE cells treated with different doses of C3a for two weeks. ( b ) Levels of TIMP-3 measured in conditioned media of the same cultures by ELISA. ( c ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with different doses of C3a for 2 weeks. ( d ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with or without C3aR antagonist for 2 weeks (ANOVA, n = 9/treatment. Data presented as mean ± SD. *p < 0.05).

Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

doi: 10.1371/journal.pone.0120411

Figure Lengend Snippet: HMVEC-d were pre-incubated with plasma of patients (N = 30) with acute HSP, plasma of healthy controls (N = 30), or culture medium alone for 48 hr, and then the cells were harvested and analyzed for the expression of C3aR (A) and CD88 (B) by flow cytometry. The expression levels were presented as mean fluorescence intensity (MFI).

Article Snippet: Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence