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: 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

Journal: bioRxiv

Article Title: B cell subsets have different capacities for phagocytosis and subsequent presentation of antigen to cognate T cells

doi: 10.1101/2025.01.28.635276

Figure Lengend Snippet: Splenic and peritoneal cavity cells were isolated from C57Bl/6 mice and incubated for 8 hrs at 37 °C and 5% CO 2 with non-cognate Ag coated beads. Some cells were pre-treated with cytochalasin D, Fc block (anti-CD16/32), anti-CD11b, or idiotype control for 1 hr at 37 °C and 5% CO 2 . Mean percentage of B cells with internalized ( A i ) IgG, ( B i ) C3b, ( C i ) C3d, or ( D ) phosphatidylserine (PS). Mean percentage of PerC B1 cells with internalized ( A ii ) IgG, ( B ii ) C3b, or ( C ii ) C3d beads. Mean percentage of T cells, macrophages, and B cells with internalized ( A iii ) IgG, ( B iii ) C3b, ( C iii ) C3d, or ( E ) OVA beads. ( F ) Immunofluorescence images of peritoneal cavity B cells from a C57/B6 mouse with associated (open arrows) or phagocytosed (closed arrows) OVA, IgG, or PS conjugated beads. Each symbol represents data from cells isolated from an individual mouse (n=4). Results from one representative of two independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 based on an ANOVA followed by a Student t test with Bonferroni correction used for multiple comparisons.

Article Snippet: Reagents for bead conjugation: Goat anti-mouse IgM F(ab’) 2 was purchased from Jackson Laboratories (115-005-020), goat anti-human IgM F(ab’) 2 from Jackson Laboratories (109-006-129), mouse C3b was purchased from CompTech (M114), mouse C3d was purchased from R&D Systems (2655-C3-050), and biotinylated PE was purchased from Cayman Chemicals (25724).

Techniques: Isolation, Incubation, Blocking Assay, Control, Immunofluorescence

Journal: bioRxiv

Article Title: B cell subsets have different capacities for phagocytosis and subsequent presentation of antigen to cognate T cells

doi: 10.1101/2025.01.28.635276

Figure Lengend Snippet: Publicly available datasets were accessed on NCBI GEO with accession numbers GSE174739, GSE232834, GSE249975 and GSE210795. B2 cells were identified as Cd19 + , Ms4a1 + , and Fcer2a + . B1 cells were identified as Cd19 + , Ms4a1 + , Bhlhe41 + , and Fcer2a - . Macrophages were identified as Adgre1 + , MERTK + , and Fcgr1A + . ( A ) Average gene expression level of various phagocytic receptors in macrophages, Fo B and B1 cells. Splenic and peritoneal cavity cells were isolated from C57Bl/6 mice and incubated for 8 hrs at 37 °C and 5% CO 2 with non-cognate Ag coated beads. ( B ) Mean percentage of B cells that were associated with (B i ) IgG, (B ii ) C3b, (B iii ) C3d, (B iv ) phosphatidylserine, or (B v ) OVA beads. Each symbol represents data from cells isolated from an individual mouse (n=4). Results from one representative of two independent experiments are shown. *p < 0.05, **p < 0.01, **** p < 0.0001 based on an ANOVA followed by a Student t test with Bonferroni correction used for multiple comparisons.

Article Snippet: Reagents for bead conjugation: Goat anti-mouse IgM F(ab’) 2 was purchased from Jackson Laboratories (115-005-020), goat anti-human IgM F(ab’) 2 from Jackson Laboratories (109-006-129), mouse C3b was purchased from CompTech (M114), mouse C3d was purchased from R&D Systems (2655-C3-050), and biotinylated PE was purchased from Cayman Chemicals (25724).

Techniques: Gene Expression, Isolation, Incubation

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 1. Anaphylatoxins C3a and C5a differentially regulate endothelial monoculture dnCI in a dose-dependent manner. (A,B) Monitoring of dnCI changes in response to 50 nM, 100 nM and 500 nM of anaphylatoxins C3a and C5a revealed a transient decrease in dnCI by 500 nM C5a after 2 h of treatment in HBMEC. At 24 h after anaphylatoxin stimulation, 500 nM C3a significantly decreased the paracellular resistance in HBMEC compared to the untreated control. (C,D) In HREC, we observed no significant alterations in paracellular resistance after 2 h of C3a or C5a treatment. A dose-dependent increase in dnCI occurred 24 h after treatment initiation in HREC treated with 50 nM C3a, 100 nM C3a, 50 nM C5a and 100 nM C5a compared to the untreated control. (A,C) ↑indicates analyses time points at 2 and 24 h. All data were normalized to the respective cell index (CI) value before the addition of treatment and to the untreated control (dnCI). (A,B) RTCA graph represents data from n = 10 for untreated and 500 nM C3a, n = 6 for 50 nM C3a and 50, 100 and 500 nM C5a and n = 8 for 100 nM C3a treated cells, respectively. (C,D) RTCA graph represents data from n = 9 for untreated, n = 6 for 50 nM C5a and 50, 100 and 500 nM C3a, n = 8 for 100 nM C5a and n = 4 for 500 nM C5a, respectively. (B,D) Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test was used for datasets including non-parametric data. One-way analysis of variance (ANOVA) with Dunnett’s T3 post hoc test was used for parametric data sets. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Control

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 2. C3a and C5a do not change CDH5 gene expression but alter VE-cadherin on protein level in HBMEC and HREC. (A,B) Treatment with 500 nM C3a or 500 nM C5a did not change CDH5 gene expression in HBMEC and HREC compared with the untreated control. (C) Two hours after treatment,

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Gene Expression, Control

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 3. Anaphylatoxin treatment caused increased C3 expression in HREC and increased C3AR1 expression in HBMEC. (A,B) qRT-PCR analysis revealed no effect of anaphylatoxin treatment on C3 expression in HBMEC but disclosed an increase in C3 expression in 500 nM C5a-treated HREC 2 h post-treatment and a 500 nM C3a and 500 nM C5a mediated increase in C3 gene expression 24 h post-treatment in HREC. (C,D) Two hours’ exposure to 500 nM C5a increased C3AR1 expression in HBMEC, while anaphylatoxin treatment had no significant effect on C3AR1 expression in HREC. (A–D) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four- hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test for datasets including non-parametric data. One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 4. C3a presence increased in HREC after exposure to C3a and C5a. (A) After 2 h, the C3 protein signal intensity increased in C3a- and C5a-treated HBMEC. The C3a signal in untreated HBMEC exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution. In HREC, C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups after 2 h, but the signal intensity increased in C3a- and C5a-treated HREC. HBMEC and HREC C3a signal intensity remained stable across treatment groups and exhibited a spot-wise pattern across all treatment groups after 2 h. (B) After 24 h, the C3 signal decreased in C3a-treated HBMEC and returned to baseline in C5a-treated cells. In untreated HBMEC, C3a exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution 24 h post-treatment initiation. The localization of C3a transitioned from spot-wise distribution to a more diffuse cellular distribution in HBMEC treated with 500 nM C3a and 500 nM C5a 24 h after treatment initiation. HREC C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups. C3 signal intensity increased in C3a- and C5a-treated HREC after 24 h. The spot-wise C3a distribution pattern seen after 2 h persisted in untreated HREC 24 h after treatment but expanded to a broader, cell-covering signal in cells treated with 500 nM C3a and 500 nMC5a. C3a signal intensity increased in C3a- and C5a-treated HREC 24 h after treatment initiation. (A,B) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 5. C5 gene and protein expression are elevated in HREC following C5a stimulation, whereas no significant changes are observed in HBMEC. (A,B) qRT-PCR indicated no variations in C5 gene expression among treatment groups or analysis time points at 2 and 24 h in HBMEC. Two hours post-treatment, exposure to 500 nM C3a and 500 nM C5a resulted in a significant upregulation in C5 gene expression in HREC. This effect was reversed 24 h post-treatment. (C,D) Stimulation with 500 nM C3a and 500 nM C5a provoked no change in C5 protein signal strength in HBMEC. Two hours post-treatment, C5 exhibited a spot-wise expression pattern in both untreated and 500 nM C3a-treated HREC, whereas HREC treated with 500 nM C5a showed a broader distribution of immunofluorescent signals. C5a increased C5 signal intensity compared to the untreated control and C3a-treated HREC. After 24 h, C5 detection signal returned to baseline in C3a- and C5a-treated cells. (A,B) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. (C,D) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Control

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 6. C5AR1 transcript expression remains unchanged, but protein detection changes under anaphylatoxic stress. (A,B) qRT-PCR analysis showed no difference in C5AR1 gene expression between untreated and C3a- or C5a-treated HBMEC and HREC. (C) Treatment with 500 nM C3a and 500 nM C5a reduced C5aR1 protein signal in HBMEC after 2 h of treatment. Immunofluorescent staining of C5aR1 in HREC showed a stable and comparable signal after 2 h in all treatment groups. (D) After 24 h, the C5aR1 signal was stable between treatment groups in HBMEC. C5aR1 protein signal intensity increased in 500 nM C5a-treated HREC after 24 h. (A,B) Data were curated from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. (C,D) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

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

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 7. Adding 50 nM of C5a, but not 100 nM or 500 nM, counteracted the barrier-disruptive effect of 500 nM C3a and improved the HBMEC barrier after 24 h. (A,B) Monitoring of combined anaphylatoxin-treated dnCI changes in HBMEC revealed a C5a dependent increase in dnCI, which counteracted the barrier-disruptive effect of C3a. 24 h post-anaphylatoxin stimulation, HBMEC treated with 500 nM C3a + 50 nM C5a reached a significantly higher dnCI compared to 500 nM C3a- treated cells. (C,D) RTCA measurement revealed an increase in dnCI in HREC treated with 500 nM C3a + 100 nM C5a 2 h after treatment addition compared to 500 nM C3a-treated cells. This regulation was maintained for 24 h after exposure to treatment. (E) Immunocytochemistry revealed a homoge- neous endothelial monolayer in all treatment groups in HBMEC, 2 h post-treatment. (F) Twenty-four hours post-treatment, 500 nM C3a decreased the VE-cadherin signal intensity, whereas treatment with 500 nM C3a + 50 nM C5a obtained a VE-cadherin signal strength similar to that of the untreated

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Immunocytochemistry

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 8. 500 nM C3a and C5a increase the transcellular permeability for IgG in HBMEC but not in HREC, reflecting reduced cell–cell contact integrity at 500 nM C3a but not enhanced paracellular

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Permeability

Journal: Theranostics

Article Title: C5aR1 is a master regulator in Colorectal Tumorigenesis via Immune modulation

doi: 10.7150/thno.45058

Figure Lengend Snippet: The strong correlation between complement activation and CRC. ( A-D ) The associations between the mRNA levels of C3 (A), C5 (B), C5AR1 (C), and C5AR2 (D) and the overall survival of CRC patients (n=364). ( E, F ) The effect of C3 , C5 , C5ar1 or C5ar2 deficiency on AOM/DSS-induced colorectal tumorigenesis compared to WT control. Images of the colorectum, where CRC often developed at distal (rectum) sites (E), and quantitative analysis of tumor number and mass (F). The experiment was duplicated. Scale bar in (E), 1 cm. ( G ) Pathological analysis of control or tumor tissues, including H&E, Ki-67 and C3d staining. Scale bar, 100 µm for H&E and 50 µm for IHC. ( H ) C5a concentration in colon tissue homogenates (CTHs) measured by ELISA. n≥ 7 in each group of WT, C3 -KO, C5 -KO, C5ar1 -KO, or C5ar2 -KO mice. Data are represented as mean ± SEM; ns, not significant; * P <0.05; and *** P <0.001. NC, negative control.

Article Snippet: Then, mouse colon sections were incubated with an affinity purified pAb specific for mouse complement component C3d (1:80; R&D Systems, Minneapolis, MN), rabbit anti-Ki67 antibody (1 μg/ml; Abcam, Cambridge, MA) or rabbit anti-CD8 alpha antibody (1:1500; Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Activation Assay, Control, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Pathogens

Article Title: Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

doi: 10.3390/pathogens14040354

Figure Lengend Snippet: Inhibition of C3b and C4b deposition by r Em CRT and its functional binding regions measured by ELISA. ( A ) Pre-incubation of 100 μL C1qD (1:50) as source of natural MBL with different amounts of r Em CRT, r Em CRT-S, or r Em CRT-NP (0, 2, 4 μM) before adding into mannan-coated plates (50 μg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b ( a ) and C4b ( b ) was detected with anti-C3 or C4 monoclonal antibodies. ( B ) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD 450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added r Em CRT).

Article Snippet: After washing with 1 × TBST containing 5 mM CaCl 2 , the C1qD diluted at 1:150 in 1 × Veronal Buffer (VB, Lonza, Basel, Switzerland) containing 0.1% gelatin, 0.05% Tween-20 was added (100 μL) as supplement of other complement components (without C1q) into each well of the plates and incubated at 37 °C for 1 h. The lectin pathway-activated C3b/C4b deposition was detected with rabbit anti-C3b monoclonal antibody (1:1000, BOSTER Biological Technology, Wuhan, China) and goat anti-C4b polyclonal antibody (1:3000, Abcam, Cambridge, UK).

Techniques: Inhibition, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay, Bioprocessing