Journal: PLoS ONE

Article Title: Human Factor H-Related Protein 2 (CFHR2) Regulates Complement Activation

doi: 10.1371/journal.pone.0078617

Figure Lengend Snippet: ( A ) CFHR2 inhibits AP activation in NHS. ( B ) CFHR2 had no effect on CP activation in NHS. TCC deposition in untreated NHS was set as 100%. C4bp is a classical pathway inhibitor. ( C ) CFHR2 and CFHR2/1–2 but not CFHR2/3–4 inhibited C3b deposition. ( D ) C3a generation in NHS activated via AP (ELISA). C3b depositon in untreated NHS was set as 100%. ( E ) CFHR2 together with factor H increased inhibition of C3b deposition in NHS as compared to CFHR2 or factor H alone. Data in ( A–E ) represent the mean values ± SD of three independent experiments ( * p≤0.05, *** p≤0.001). Control (ctrl) indicates binding of the detection Ab to the plate.

Article Snippet: For comparison CFHR2, factor H and CRIg (each 0.001–1 μM) were added to NHS (20% in HEPES-EGTA buffer) incubated with LPS coated wells as described and complement activation was followed by C3b deposition and detection of C3a or Ba in the supernatant by ELISA Kits (Quidel).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Control, Binding Assay

Journal: PLoS ONE

Article Title: Human Factor H-Related Protein 2 (CFHR2) Regulates Complement Activation

doi: 10.1371/journal.pone.0078617

Figure Lengend Snippet: CFHR2 inhibited in vitro assembled AP C3 convertase as measured by C3a generation. The corresponding densitometric analysis of the C3a bands of the Western blot is shown. C3a generation by C3 convertase alone was normalized to 100%. A representative analysis of three experiments is shown.

Article Snippet: For comparison CFHR2, factor H and CRIg (each 0.001–1 μM) were added to NHS (20% in HEPES-EGTA buffer) incubated with LPS coated wells as described and complement activation was followed by C3b deposition and detection of C3a or Ba in the supernatant by ELISA Kits (Quidel).

Techniques: In Vitro, Western Blot

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) RT-PCR analysis of total versican in tubular cells treated with 20% patient serum (PS), 20% heat-inactivated patient serum (HIPS), 20% healthy serum (HS), or 20% heat-inactivated healthy serum (HIHS) for 48 hours (n = 3). (B) RT-PCR analysis of total versican in tubular cells treated with 20% PS, eculizumab, or SB290157 for 48 hours (n = 5). (C) Level of serum C3a in FSGS patients and controls (n = 20). (D) Level of urinary C3a in FSGS patients and controls (n = 20). (E) Immunofluorescence staining of C3a (red) and C3aR (green) in renal tissue of FSGS patients (n = 3). (F) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with C3a or 20% PS for 48 hours (n = 3). Scale bars: 20 μm. For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for A, B, and F, and a 2-tailed Student’s t test was used for C and D. *P < 0.05 compared with control; #P < 0.05 compared with PS-, HS-, or C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) RT-PCR analysis of total versican in tubular cells treated with C3a and SB203580, PD098059, or MK2206 (n = 5). (B) RT-PCR analysis of versican V1, V0, and V3 in tubular cells treated with C3a and MK2206 (n = 3). (C) Western blot analysis of phospho-AKT (p-AKT) in tubular cells (n = 3). (D) Western blot analysis of nuclear β-catenin in tubular cells (n = 3). (E) Schematic of the β-catenin/TCF binding sites in the upstream sequence of the versican promoter and the constructed versican promoter–luciferase reporter plasmids. (F) ChIP analysis of the binding between β-catenin and the versican promoter in tubular cells treated with C3a (n = 3). (G) Normalized luciferase activity of reporter constructs in tubular cells cotransfected with N90-β-catenin plasmid (n = 3). (H) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells transfected with N90-β-catenin plasmid (n = 3). (I) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with C3a and si-CTNNB1 (n = 3). For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for A, B, and I, and a 2-tailed Student’s t test was used for G and H. *P < 0.05 compared with control; #P < 0.05 compared with C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Sequencing, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Level of serum creatinine in WT and C3aR-knockout (C3aR–/–) mice treated with Adriamycin (ADR) (n = 6). (B and C) Masson’s trichrome staining of renal sections in WT and C3aR–/– mice treated with ADR (n = 6). (D) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubulointerstitial tissues of WT and C3aR–/– mice treated with ADR (n = 6). (E and F) Immunohistochemical staining of versican in renal tissues of WT and C3aR–/– mice treated with ADR (n = 6). (G) Level of urinary C3a in WT and C3aR–/– mice treated with ADR (n = 6). (H) Immunofluorescence staining of C3a in WT and C3aR–/– mice treated with ADR (n = 6). (I) Western blot analysis of p-AKT in tubulointerstitial tissues of WT and C3aR–/– mice treated with ADR (n = 6). (J) Western blot analysis of nuclear β-catenin (n = 6). (K) ChIP analysis of the binding between β-catenin and the versican promoter in tubulointerstitial tissues (n = 6). (L) Western blot analysis of versican V1 in tubulointerstitial tissues (n = 6). Scale bars: 20 μm (E and H). Scale bars: 30 μm (B). For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for A, C, D, F, and G. *P < 0.05 compared with control WT mice; #P < 0.05 compared with ADR-treated WT mice.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Knock-Out, Staining, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Immunofluorescence, Western Blot, Binding Assay, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Level of serum suPAR in FSGS patients (n = 20). (B) Level of urinary suPAR in FSGS patients (n = 20). (C) Immunohistochemical staining of uPAR in renal tissues of FSGS patients (n = 5). (D) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and uPAR-blocking antibody (n = 5). (E) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with C3a and suPAR (n = 5). (F) RT-PCR analysis of ITGB6 in tubulointerstitial tissues of FSGS patients (n = 20). (G) IP analysis of the binding between suPAR and ITGB6 in tubular cells treated with 20% PS (n = 3). (H) Immunofluorescence staining of uPAR (green) and ITGB6 (red) in tubular cells treated with 20% PS (n = 3). Scale bars: 20 μm (C and H). (I) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and si-ITGB6 (n = 5). (J) Rac1 activation assay in tubular cells treated with 20% PS and si-ITGB6 (n = 3). (K) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and si-Rac1 (n = 5). For statistical analysis, a 2-tailed Student’s t test was used for A, B, and F, and 1-way ANOVA with Tukey’s post hoc test was used for D, E, I, J, and K. *P < 0.05 compared with control; #P < 0.05 compared with PS- or C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Binding Assay, Immunofluorescence, Activation Assay, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Splicing factors predicted to bind to the 5′ end of versican exon 7 with the SpliceAid 2 database. (B) IP analysis of the binding between Rac1 and SRp40 in tubular cells treated with 20% PS (n = 3). (C) Immunofluorescence staining of SRp40 (green), Rac1 (red), and DAPI (blue) in tubular cells treated with 20% PS (n = 3). Scale bars: 20 μm. (D) RIP analysis of the binding of SRp40 and Rac1 to the 5′ end of versican exon 7 in tubular cells treated with 20% PS (n = 5). (E) RIP analysis of the binding of Rac1 to the 5′ end of versican exon 7 in tubular cells treated with 20% PS and si-SRp40 (n = 3). (F) RIP analysis of the binding of SRp40 to the 5′ end of versican exon 7 in tubular cells treated with 20% PS and si-Rac1 (n = 3). (G) Rac1 activity in tubular cells treated with 20% PS and si-SRp40 (n = 3). (H) RIP analysis of the binding of U2AF1 to the 3′ splice site of versican intron 6 in tubular cells treated with 20% PS and pGEMT-SRp40 plasmid or suPAR-blocking antibody (n = 5). (I) PCR analysis of versican V0 and V1 in tubular cells treated with 20% PS and pGEMT-SRp40 plasmid or suPAR-blocking antibody (n = 3). (J) RIP analysis of the binding of U2AF1 to the 3′ splice site of versican intron 6 in tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 5). (K) PCR analysis of versican V0 and V1 in tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 3). For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for D–F, G, H, and J. *P < 0.05 compared with control; #P < 0.05 compared with PS- or C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Binding Assay, Immunofluorescence, Staining, Activity Assay, Plasmid Preparation, Blocking Assay, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Model of the 34-nt complementary sequence in intron 6 (site 1) and intron 8 (site 2) of versican pre-mRNA. (B) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells (n = 3). (C) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells treated with LNA antisense oligonucleotides targeting either site 1 or site 2 (n = 3). (D) PCR analysis of versican V3 in tubular cells (n = 3). (E) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells transfected with Rac1Q61L plasmid and si-SRp40 (n = 3). (F) PCR analysis of versican V3 in tubular cells transfected with Rac1Q61L plasmid and si-SRp40 (n = 3). (G) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells treated with 20% PS and si-Rac1 or suPAR-blocking antibody (n = 3). (H) PCR analysis of versican V3 in tubular cells treated with 20% PS and si-Rac1 or suPAR-blocking antibody (n = 3). (I) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 3). (J) PCR analysis of versican V3 in tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 3).

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Sequencing, Transfection, Plasmid Preparation, Blocking Assay

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: C3a promotes the transcription of versican by activating the AKT/β-catenin pathway in tubular cells. suPAR binds to ITGFB6 and activates Rac1, which translocates into the nucleus and binds to SRp40 at the 5′ end of exon 7 of versican pre-mRNA. This binding not only inhibits the 3′-end splicing of intron 6 but also inhibits the base-pair interaction between intron 6 and intron 8, which leads to the formation of versican V1.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Binding Assay

Journal: Anesthesia & Analgesia

Article Title: Analgesic Effect of Exercise on Neuropathic Pain via Regulating the Complement Component 3 of Reactive Astrocytes

doi: 10.1213/ane.0000000000006884

Figure Lengend Snippet: Figure 5. Effect of C3 on exercise-induced analgesic effect. A and B, The dose-dependent effect of rC3 (i.t.) on mechanical (A) and cold (B) pain behaviors. One-way ANOVA was used to test the statistical difference and the post hoc Bonferroni test indicates that 50 ng rC3 induced both mechanical (** P = .0062) and cold pain behaviors (** P = .0028). C–E, Analgesic effect of exercise on rC3-induced mechanical and cold allodynia. Schematic of experimental approach (C) and bar graph (D to E) showing significantly higher PWT (* P = .0168) and decreased cold pain behavior (** P = .0025) in the rC3 combined exercise group than rC3 alone group. F–H, The effect of subeffective dose of rC3 on pain threshold of SNI mice after 2 weeks training. Schematic of experimental approach (F) and the changes of PWT (* P = .0348) (G) and cold sensitivity (* P = .0191) (H) were significant after 10 ng rC3 administration; n=7–8 per group. D–H, 2-way ANOVA followed by Bonferroni post hoc test was used to analyze the statistical difference. Data are represented as mean ± SEM. ANOVA indicates analysis of variance; PWT, paw withdrawal threshold; rC3, recombinant C3; SEM, standard error of the mean; SNI, spared nerve injury.

Article Snippet: Intrathecal Injection Intrathecal injections were performed under isoflurane anesthesia as described above.14 10 μL recombinant mouse C3 (rC3, R&D Systems Inc, 8085) or phosphate-buffered saline (PBS) solution was injected into intervertebral space by using a 10-μL microinjection syringe as previously described.

Techniques: Recombinant

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Microfluidic devices for measuring neutrophil chemotaxis, phagocytosis, and swarming behaviors. (A) Microscopic image of a segment of the chemotaxis microfluidic device (x10 Brightfield, Nikon TiE) showing neutrophil chemotaxis towards C5a and C3a during increasing mechanical restriction. Each chemoattractant chamber is connected to the cell loading channel through 3 tapered channels. (B) Microscopic image of a segment of the phagocytosis microfluidic device (x10 Brightfield/Fluorescent, Nikon TiE) showing neutrophils (blue nuclei) from the outer chamber migrating towards the central reservoir through a migration channel to phagocytose S. aureus particles (green) in plasma at T 20 minutes. (C) Microscopic images obtained at three different time points (T 0 , T 10 , and T 30 minutes) showing the formation of a neutrophil swarm (blue nuclei) over a cluster of Texas red-labeled zymosan A S. cerevisiae bioparticles (Red).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Migration, Clinical Proteomics, Labeling

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Chemotaxis of isolated neutrophils towards C5a and C3a and the effect of AVA on chemotaxis. (A) The percentage of neutrophils migrating directionally towards increasing concentrations of C5a and C3a compared to media alone (HBSS with 0.5%BSA, N=3, ****p < 0.0001; One-way ANOVA with Dunnett’s test) (B) C3a inhibits neutrophil chemotaxis towards C5a, C5a effect is dependent on concentration, and there is no interaction effect between C3 and C5a. (N=3 donors, p < 0.005, Repeated measures, two way ANOVA, with Sidak’s correction for multiple comparisons). (C) Percentage of neutrophils migrating directionally towards C5a (100 nM) after treatment of neutrophils with C5aR1 antagonist AVA (N=3, 4 or 5, each dot on the plot represents one experiment, ****p < 0.01 for comparisons to C5a control; ns represents not statistical significant. One way ANOVA with Dunnett’s multiple comparison test with single pooled variance).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Isolation, Concentration Assay, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Atypical membrane elasticity of neutrophils and migration patterns when exposed to high C5a dose. (A) Percentage of neutrophil migration patterns during chemotaxis towards C5a, C3a, and fMLP. (N=3). (B) Microscopic images of elongated neutrophils when exposed to C5a 1µM inside the end chambers. Scale = 100 µm. (C) Percentages of unique reversed migration phenotype of neutrophils observed when exposed to high C5a concentration. (N=3, *p < 0.05; ns represents not statistical significant. One-way ANOVA with Dunnett’s test).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Membrane, Migration, Chemotaxis Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Effect of C5 complement inhibitors on neutrophil chemotaxis towards endogenous complement activation with CVF. (A) Effect of AVA (250 nM) on endogenous complement activation by increasing CVF doses (N=3 donors, *p < 0.05, **p < 0.01; paired two-tailed t -test). (B) Effect of C5 inhibition with ECU (3 µM) and RA101295 (3 µM) on endogenous complement activation by CVF (N=3, **p < 0.01; ns represents not statistical significant. One-way ANOVA with Dunnett’s test).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Activation Assay, Two Tailed Test, Inhibition

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Schematic representation of complement activation pathways, terminal inhibition strategies, and their effects on neutrophil functional behaviors. The classical, lectin, and alternative pathways converge on the cleavage of the central components C3 into C3a and C3b peptides. C3a acts as an anti-inflammatory mediator towards neutrophils, while C3b enhances phagocytosis through opsonization and forms C5 convertases, which cleaves C5 into C5a and C5b. C5a is a potent pro-inflammatory mediator and chemoattractant, while C5b forms the membrane attack complex (MAC), leading to cell and bacteria lysis. ECU and RA101295 are C5-inhibitors, which block the production of C5a and C5b peptides, while AVA blocks C5a mediated chemotaxis and pro-inflammatory effects.

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Activation Assay, Inhibition, Functional Assay, Membrane, Bacteria, Lysis, Blocking Assay, Chemotaxis Assay