Review

recombinant human c5a rhc5a (MedChemExpress)

MedChemExpress is a verified supplier

MedChemExpress manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

MedChemExpress recombinant human c5a rhc5a

TNF primes neutrophils and an allosteric FFA2R modulator turns <t>C5a</t> into a more potent activator of the NADPH oxidase. For priming, neutrophils were incubated with TNF (10 ng/mL) at 37 °C for 20 min and then stored on ice until used. The NADPH oxidase activity (O 2 - production) induced by C5a was measured continuously and expressed in mega couts per minute (Mcpm). (A) The response induced in naïve (non-primed; dashed line) and TNF primed neutrophils (solid line) by C5a (2 nM, added at the time point marked with an arrow) was measured. Inset: The priming effect of TNF on the NADPH oxidase activity expressed as peak values (Mcpm; mean ± SEM) of O 2 - production induced by C5a in naïve (n = 5) and TNF-primed neutrophils (n = 14). (B) Inhibition of the C5a-induced response by the specific C5aR1 antagonist avacopan. TNF-primed neutrophils were incubated without (solid line) or with avacopan (50 nM, dashed line) for 5 min before addition of C5a (2 nM; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The inhibitory effect of avacopan on the neutrophil NADPH oxidase activity expressed as peak values (Mcpm) of O 2 - production induced by C5a in absence and presence of the antagonist, respectively (mean ± SEM, n = 6). (C) Effects of the allosteric FFA2R modulator Cmp58 on the response induced by different concentrations of C5a in TNF-primed neutrophils. Neutrophils were incubated without (dashed lines) or with Cmp58 (1 µM, solid lines) for 5 min before addition of C5a (0.1 nM, grey lines, or 2 nM, black lines; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The effect of Cmp58 on the NADPH oxidase activity expressed as fold increase of O 2 - production induced by different C5a concentrations (0.1, 0.25 or 2 nM) in the absence and presence of Cmp58, respectively (mean ± SEM, n = 6). The horizontal dotted line in the bar graph represents a ratio of 1. (D) TNF-primed neutrophils were pre-incubated with and without Cmp58 (1 µM) for 5 min and activated with different concentrations of C5a as indicated. Superoxide production was recorded continuously and expressed as the peak value obtained in terms of percent of the activity induced by C5a alone (2 nM, mean ± SEM, n = 3).One representative experiment is shown in each subset (A–C) . Statistically significant differences in the insets were evaluated by an unpaired Student’s t -test (A) , a paired Student’s t -test (B) , or a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) and are denoted as *( p ≤ 0.05), ***( p ≤ 0.001), and ns = not significant.

Recombinant Human C5a Rhc5a, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+c5a/Complement+C5%2C+Human/pmc13012905-42-0-19
Average 93 stars, based on 1 article reviews

recombinant human c5a rhc5a - by Bioz Stars, 2026-09

93/100 stars

Images

1) Product Images from "The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan"

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2026.1795719

TNF primes neutrophils and an allosteric FFA2R modulator turns C5a into a more potent activator of the NADPH oxidase. For priming, neutrophils were incubated with TNF (10 ng/mL) at 37 °C for 20 min and then stored on ice until used. The NADPH oxidase activity (O 2 - production) induced by C5a was measured continuously and expressed in mega couts per minute (Mcpm). (A) The response induced in naïve (non-primed; dashed line) and TNF primed neutrophils (solid line) by C5a (2 nM, added at the time point marked with an arrow) was measured. Inset: The priming effect of TNF on the NADPH oxidase activity expressed as peak values (Mcpm; mean ± SEM) of O 2 - production induced by C5a in naïve (n = 5) and TNF-primed neutrophils (n = 14). (B) Inhibition of the C5a-induced response by the specific C5aR1 antagonist avacopan. TNF-primed neutrophils were incubated without (solid line) or with avacopan (50 nM, dashed line) for 5 min before addition of C5a (2 nM; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The inhibitory effect of avacopan on the neutrophil NADPH oxidase activity expressed as peak values (Mcpm) of O 2 - production induced by C5a in absence and presence of the antagonist, respectively (mean ± SEM, n = 6). (C) Effects of the allosteric FFA2R modulator Cmp58 on the response induced by different concentrations of C5a in TNF-primed neutrophils. Neutrophils were incubated without (dashed lines) or with Cmp58 (1 µM, solid lines) for 5 min before addition of C5a (0.1 nM, grey lines, or 2 nM, black lines; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The effect of Cmp58 on the NADPH oxidase activity expressed as fold increase of O 2 - production induced by different C5a concentrations (0.1, 0.25 or 2 nM) in the absence and presence of Cmp58, respectively (mean ± SEM, n = 6). The horizontal dotted line in the bar graph represents a ratio of 1. (D) TNF-primed neutrophils were pre-incubated with and without Cmp58 (1 µM) for 5 min and activated with different concentrations of C5a as indicated. Superoxide production was recorded continuously and expressed as the peak value obtained in terms of percent of the activity induced by C5a alone (2 nM, mean ± SEM, n = 3).One representative experiment is shown in each subset (A–C) . Statistically significant differences in the insets were evaluated by an unpaired Student’s t -test (A) , a paired Student’s t -test (B) , or a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) and are denoted as *( p ≤ 0.05), ***( p ≤ 0.001), and ns = not significant.

Figure Legend Snippet: TNF primes neutrophils and an allosteric FFA2R modulator turns C5a into a more potent activator of the NADPH oxidase. For priming, neutrophils were incubated with TNF (10 ng/mL) at 37 °C for 20 min and then stored on ice until used. The NADPH oxidase activity (O 2 - production) induced by C5a was measured continuously and expressed in mega couts per minute (Mcpm). (A) The response induced in naïve (non-primed; dashed line) and TNF primed neutrophils (solid line) by C5a (2 nM, added at the time point marked with an arrow) was measured. Inset: The priming effect of TNF on the NADPH oxidase activity expressed as peak values (Mcpm; mean ± SEM) of O 2 - production induced by C5a in naïve (n = 5) and TNF-primed neutrophils (n = 14). (B) Inhibition of the C5a-induced response by the specific C5aR1 antagonist avacopan. TNF-primed neutrophils were incubated without (solid line) or with avacopan (50 nM, dashed line) for 5 min before addition of C5a (2 nM; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The inhibitory effect of avacopan on the neutrophil NADPH oxidase activity expressed as peak values (Mcpm) of O 2 - production induced by C5a in absence and presence of the antagonist, respectively (mean ± SEM, n = 6). (C) Effects of the allosteric FFA2R modulator Cmp58 on the response induced by different concentrations of C5a in TNF-primed neutrophils. Neutrophils were incubated without (dashed lines) or with Cmp58 (1 µM, solid lines) for 5 min before addition of C5a (0.1 nM, grey lines, or 2 nM, black lines; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The effect of Cmp58 on the NADPH oxidase activity expressed as fold increase of O 2 - production induced by different C5a concentrations (0.1, 0.25 or 2 nM) in the absence and presence of Cmp58, respectively (mean ± SEM, n = 6). The horizontal dotted line in the bar graph represents a ratio of 1. (D) TNF-primed neutrophils were pre-incubated with and without Cmp58 (1 µM) for 5 min and activated with different concentrations of C5a as indicated. Superoxide production was recorded continuously and expressed as the peak value obtained in terms of percent of the activity induced by C5a alone (2 nM, mean ± SEM, n = 3).One representative experiment is shown in each subset (A–C) . Statistically significant differences in the insets were evaluated by an unpaired Student’s t -test (A) , a paired Student’s t -test (B) , or a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) and are denoted as *( p ≤ 0.05), ***( p ≤ 0.001), and ns = not significant.

Techniques Used: Incubation, Activity Assay, Inhibition, Comparison

Antagonists selective for FFA2R and C5aR1, respectively, have inhibiting profiles beyond the expected receptor specificity. TNF-primed neutrophils were incubated with Cmp58 (1 µM; 5 min at 37 °C) and to determine the inhibitory effects of receptor specific antagonist, these cells were incubated without or with an antagonist (CATPB, 100 nM; avacopan, 50 nM) and the O 2 - production was measured continuously following an activation by different concentrations of C5a. (A-C) Inhibition by the antagonists (CATPB and avacopan) of the response in neutrophils incubated with Cmp58 and induced by C5a, at different concentrations, i.e., 2 nM (A; n = 3-8), 0.25 nM (B; n = 7), and 0.1 nM (C; n = 6), respectively. Inhibition is expressed as the remaining activity (peak value in percent) of neutrophils after activation with C5a in the presence of the respective antagonist. Bar graphs are presented as mean ± SEM. The statistical determinations are based on the difference between the C5a response without and with the antagonists. Statistically significant differences were evaluated by a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) or by a mixed effects analysis followed by Šίdák’s multiple comparison (A, B) and are denoted as *( p ≤ 0.05), **( p ≤ 0.01), ***( p ≤ 0.001), and ns = not significant.

Figure Legend Snippet: Antagonists selective for FFA2R and C5aR1, respectively, have inhibiting profiles beyond the expected receptor specificity. TNF-primed neutrophils were incubated with Cmp58 (1 µM; 5 min at 37 °C) and to determine the inhibitory effects of receptor specific antagonist, these cells were incubated without or with an antagonist (CATPB, 100 nM; avacopan, 50 nM) and the O 2 - production was measured continuously following an activation by different concentrations of C5a. (A-C) Inhibition by the antagonists (CATPB and avacopan) of the response in neutrophils incubated with Cmp58 and induced by C5a, at different concentrations, i.e., 2 nM (A; n = 3-8), 0.25 nM (B; n = 7), and 0.1 nM (C; n = 6), respectively. Inhibition is expressed as the remaining activity (peak value in percent) of neutrophils after activation with C5a in the presence of the respective antagonist. Bar graphs are presented as mean ± SEM. The statistical determinations are based on the difference between the C5a response without and with the antagonists. Statistically significant differences were evaluated by a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) or by a mixed effects analysis followed by Šίdák’s multiple comparison (A, B) and are denoted as *( p ≤ 0.05), **( p ≤ 0.01), ***( p ≤ 0.001), and ns = not significant.

Techniques Used: Incubation, Activation Assay, Inhibition, Activity Assay, Comparison

Differential NADPH oxidase activity triggered by Cmp58 in neutrophils pretreated with C5a or propionate. (A) TNF-primed neutrophils pre-incubated with Cmp58 (1 µM, 5 min at 37 °C) were activated by C5a (0.1 nM, solid line) or propionate (25 µM, dashed line). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by the ligands C5a and propionate, in neutrophils pre-incubated (abbreviation Pre-inc.) with Cmp58, expressed as the peak values of O 2 - production (Mcpm; mean ± SEM, n = 5). (B) TNF-primed neutrophils incubated for 5 min at 37 °C with non-activating concentrations of C5a (solid line; 0.1 nM) or propionate (dashed line; 25 µM) were activated by Cmp58 (1 µM). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by Cmp58, in neutrophils pre-incubated (abbreviation Pre-inc.) with C5a and propionate, respectively, expressed as the peak values of O 2 - production (mean ± SEM, n = 5). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05) and ns, not significant.

Figure Legend Snippet: Differential NADPH oxidase activity triggered by Cmp58 in neutrophils pretreated with C5a or propionate. (A) TNF-primed neutrophils pre-incubated with Cmp58 (1 µM, 5 min at 37 °C) were activated by C5a (0.1 nM, solid line) or propionate (25 µM, dashed line). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by the ligands C5a and propionate, in neutrophils pre-incubated (abbreviation Pre-inc.) with Cmp58, expressed as the peak values of O 2 - production (Mcpm; mean ± SEM, n = 5). (B) TNF-primed neutrophils incubated for 5 min at 37 °C with non-activating concentrations of C5a (solid line; 0.1 nM) or propionate (dashed line; 25 µM) were activated by Cmp58 (1 µM). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by Cmp58, in neutrophils pre-incubated (abbreviation Pre-inc.) with C5a and propionate, respectively, expressed as the peak values of O 2 - production (mean ± SEM, n = 5). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05) and ns, not significant.

Techniques Used: Activity Assay, Incubation

C5a triggers a transient increase in the cytosolic concentration of free calcium ions ([Ca 2+ ] i ) in neutrophils, which is not affected by the allosteric FFA2R modulator Cmp58. Fura-2 loaded neutrophils were activated by propionate (25 µM) or different concentrations of C5a (2, 0.25, 0.1, and 0.05 nM) in the absence (upper panel) or presence (lower panel) of the allosteric FFA2R modulator Cmp58 (1 µM, pre-incubated with the cells for 10 min at 37 °C prior addition of the agonist). Results obtained are shown as one representative experiment out of 3. The arrows show the time point for the addition of the agonists to the Fura-2 labelled neutrophils.

Figure Legend Snippet: C5a triggers a transient increase in the cytosolic concentration of free calcium ions ([Ca 2+ ] i ) in neutrophils, which is not affected by the allosteric FFA2R modulator Cmp58. Fura-2 loaded neutrophils were activated by propionate (25 µM) or different concentrations of C5a (2, 0.25, 0.1, and 0.05 nM) in the absence (upper panel) or presence (lower panel) of the allosteric FFA2R modulator Cmp58 (1 µM, pre-incubated with the cells for 10 min at 37 °C prior addition of the agonist). Results obtained are shown as one representative experiment out of 3. The arrows show the time point for the addition of the agonists to the Fura-2 labelled neutrophils.

Techniques Used: Concentration Assay, Incubation

Activation of FFA2R inhibits (heterologously desensitizes) the neutrophil response induced by C5a. TNF-primed neutrophils incubated with Cmp58 (1 µM), were either left in a resting state (dashed lines) or activated by an FFA2R activating/transactivating ligand (solid lines) and the O 2 - production was measured continuously and expressed in Mcpm. When the response induced by the FFA2R activating agonist was terminated, the two cell samples were activated by an addition of C5a (2 nM). The results obtained in one representative experiment is shown together with an inset showing the peak activities induced by C5a (mean ± SEM, n = 3). (A) Propionate (25 µM), (B) ATP (25µM), and (C) AZ1729 (1µM) were used as the FFA2R activating ligand (the time point for addition is marked by arrow 1). The time point for addition of C5a to the cells is marked by arrow 2. Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05).

Figure Legend Snippet: Activation of FFA2R inhibits (heterologously desensitizes) the neutrophil response induced by C5a. TNF-primed neutrophils incubated with Cmp58 (1 µM), were either left in a resting state (dashed lines) or activated by an FFA2R activating/transactivating ligand (solid lines) and the O 2 - production was measured continuously and expressed in Mcpm. When the response induced by the FFA2R activating agonist was terminated, the two cell samples were activated by an addition of C5a (2 nM). The results obtained in one representative experiment is shown together with an inset showing the peak activities induced by C5a (mean ± SEM, n = 3). (A) Propionate (25 µM), (B) ATP (25µM), and (C) AZ1729 (1µM) were used as the FFA2R activating ligand (the time point for addition is marked by arrow 1). The time point for addition of C5a to the cells is marked by arrow 2. Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05).

Techniques Used: Activation Assay, Incubation

C5a-induced desensitization in Cmp58-sensitized neutrophils. (A–C) TNF-primed neutrophils incubated with Cmp58 (1 µM, 5 min at 37 °C) were either left in a resting state (dashed lines) or activated by C5a (2 nM, solid line; the time point for addition is marked by arrow 1), and the O 2 - production was measured continuously and expressed as Mcpm. When the response, induced by the C5aR1 activating ligand was terminated, the two cell samples were activated by the addition of (A) propionate (25 µM, an FFA2R activating ligand), (B) ATP (50 µM, a P2Y 2 R activating ligand), or (C) AZ1729 (1 µM, an FFA2R activating ligand), and the time point for their addition is marked by arrow 2 (D–F) . The experimental setup differs from that described above, in that Cmp58 (1 µM), was added first when the response induced by C5a (time for addition marked by arrow 1), had settled. The neutrophils were then activated with either propionate [ (D) ; 25 µM)], ATP [ (E) ; 50 µM)], or AZ1729 [ (F) ; 1 µM)]. The time point for addition of the FFA2R activating/transactivating ligand is marked by arrow 2, and one representative experiment is depicted. The results obtained are shown together with an inset showing the peak activities induced by the FFA2R activating/transactivating ligands (mean ± SEM, n = 3). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as * ( p ≤ 0.05) and ns = not significant.

Figure Legend Snippet: C5a-induced desensitization in Cmp58-sensitized neutrophils. (A–C) TNF-primed neutrophils incubated with Cmp58 (1 µM, 5 min at 37 °C) were either left in a resting state (dashed lines) or activated by C5a (2 nM, solid line; the time point for addition is marked by arrow 1), and the O 2 - production was measured continuously and expressed as Mcpm. When the response, induced by the C5aR1 activating ligand was terminated, the two cell samples were activated by the addition of (A) propionate (25 µM, an FFA2R activating ligand), (B) ATP (50 µM, a P2Y 2 R activating ligand), or (C) AZ1729 (1 µM, an FFA2R activating ligand), and the time point for their addition is marked by arrow 2 (D–F) . The experimental setup differs from that described above, in that Cmp58 (1 µM), was added first when the response induced by C5a (time for addition marked by arrow 1), had settled. The neutrophils were then activated with either propionate [ (D) ; 25 µM)], ATP [ (E) ; 50 µM)], or AZ1729 [ (F) ; 1 µM)]. The time point for addition of the FFA2R activating/transactivating ligand is marked by arrow 2, and one representative experiment is depicted. The results obtained are shown together with an inset showing the peak activities induced by the FFA2R activating/transactivating ligands (mean ± SEM, n = 3). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as * ( p ≤ 0.05) and ns = not significant.

Techniques Used: Incubation

A proposed model for how the neutrophil NADPH oxidase is turned to an active state by the C5aR1 agonist C5a in the absence and presence of the allosteric FFA2R modulator Cmp58. (A) Left: The neutrophil response induced by a 2 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating NADPH oxidase and have also the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating oxidase and transactivate the allosterically modulated FFA2R; the transactivated FFA2R does not directly potentiate the NADPH oxidase response but reduces the inhibitory effect on this response, of the C5aR1 specific antagonist avacopan. (B) Left: The neutrophil response induced by a 0.1 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 have no NADPH oxidase activating effect. The signals have, however, the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 activate the O 2 -- generating NADPH oxidase, and this activation is totally dependent of the signals generated by the allosterically modulated FFA2R, a receptor made receptive to the transactivating signals generated by C5aR1.

Figure Legend Snippet: A proposed model for how the neutrophil NADPH oxidase is turned to an active state by the C5aR1 agonist C5a in the absence and presence of the allosteric FFA2R modulator Cmp58. (A) Left: The neutrophil response induced by a 2 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating NADPH oxidase and have also the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating oxidase and transactivate the allosterically modulated FFA2R; the transactivated FFA2R does not directly potentiate the NADPH oxidase response but reduces the inhibitory effect on this response, of the C5aR1 specific antagonist avacopan. (B) Left: The neutrophil response induced by a 0.1 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 have no NADPH oxidase activating effect. The signals have, however, the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 activate the O 2 -- generating NADPH oxidase, and this activation is totally dependent of the signals generated by the allosterically modulated FFA2R, a receptor made receptive to the transactivating signals generated by C5aR1.

Techniques Used: Concentration Assay, Generated, Activation Assay

Isolation:

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade
Article Snippet: .. PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark. ..

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade.
Article Snippet: .. PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark. ..

Incubation:

Image Search Results

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: TNF primes neutrophils and an allosteric FFA2R modulator turns C5a into a more potent activator of the NADPH oxidase. For priming, neutrophils were incubated with TNF (10 ng/mL) at 37 °C for 20 min and then stored on ice until used. The NADPH oxidase activity (O 2 - production) induced by C5a was measured continuously and expressed in mega couts per minute (Mcpm). (A) The response induced in naïve (non-primed; dashed line) and TNF primed neutrophils (solid line) by C5a (2 nM, added at the time point marked with an arrow) was measured. Inset: The priming effect of TNF on the NADPH oxidase activity expressed as peak values (Mcpm; mean ± SEM) of O 2 - production induced by C5a in naïve (n = 5) and TNF-primed neutrophils (n = 14). (B) Inhibition of the C5a-induced response by the specific C5aR1 antagonist avacopan. TNF-primed neutrophils were incubated without (solid line) or with avacopan (50 nM, dashed line) for 5 min before addition of C5a (2 nM; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The inhibitory effect of avacopan on the neutrophil NADPH oxidase activity expressed as peak values (Mcpm) of O 2 - production induced by C5a in absence and presence of the antagonist, respectively (mean ± SEM, n = 6). (C) Effects of the allosteric FFA2R modulator Cmp58 on the response induced by different concentrations of C5a in TNF-primed neutrophils. Neutrophils were incubated without (dashed lines) or with Cmp58 (1 µM, solid lines) for 5 min before addition of C5a (0.1 nM, grey lines, or 2 nM, black lines; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The effect of Cmp58 on the NADPH oxidase activity expressed as fold increase of O 2 - production induced by different C5a concentrations (0.1, 0.25 or 2 nM) in the absence and presence of Cmp58, respectively (mean ± SEM, n = 6). The horizontal dotted line in the bar graph represents a ratio of 1. (D) TNF-primed neutrophils were pre-incubated with and without Cmp58 (1 µM) for 5 min and activated with different concentrations of C5a as indicated. Superoxide production was recorded continuously and expressed as the peak value obtained in terms of percent of the activity induced by C5a alone (2 nM, mean ± SEM, n = 3).One representative experiment is shown in each subset (A–C) . Statistically significant differences in the insets were evaluated by an unpaired Student’s t -test (A) , a paired Student’s t -test (B) , or a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) and are denoted as *( p ≤ 0.05), ***( p ≤ 0.001), and ns = not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Incubation, Activity Assay, Inhibition, Comparison

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: Antagonists selective for FFA2R and C5aR1, respectively, have inhibiting profiles beyond the expected receptor specificity. TNF-primed neutrophils were incubated with Cmp58 (1 µM; 5 min at 37 °C) and to determine the inhibitory effects of receptor specific antagonist, these cells were incubated without or with an antagonist (CATPB, 100 nM; avacopan, 50 nM) and the O 2 - production was measured continuously following an activation by different concentrations of C5a. (A-C) Inhibition by the antagonists (CATPB and avacopan) of the response in neutrophils incubated with Cmp58 and induced by C5a, at different concentrations, i.e., 2 nM (A; n = 3-8), 0.25 nM (B; n = 7), and 0.1 nM (C; n = 6), respectively. Inhibition is expressed as the remaining activity (peak value in percent) of neutrophils after activation with C5a in the presence of the respective antagonist. Bar graphs are presented as mean ± SEM. The statistical determinations are based on the difference between the C5a response without and with the antagonists. Statistically significant differences were evaluated by a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) or by a mixed effects analysis followed by Šίdák’s multiple comparison (A, B) and are denoted as *( p ≤ 0.05), **( p ≤ 0.01), ***( p ≤ 0.001), and ns = not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Incubation, Activation Assay, Inhibition, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: Differential NADPH oxidase activity triggered by Cmp58 in neutrophils pretreated with C5a or propionate. (A) TNF-primed neutrophils pre-incubated with Cmp58 (1 µM, 5 min at 37 °C) were activated by C5a (0.1 nM, solid line) or propionate (25 µM, dashed line). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by the ligands C5a and propionate, in neutrophils pre-incubated (abbreviation Pre-inc.) with Cmp58, expressed as the peak values of O 2 - production (Mcpm; mean ± SEM, n = 5). (B) TNF-primed neutrophils incubated for 5 min at 37 °C with non-activating concentrations of C5a (solid line; 0.1 nM) or propionate (dashed line; 25 µM) were activated by Cmp58 (1 µM). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by Cmp58, in neutrophils pre-incubated (abbreviation Pre-inc.) with C5a and propionate, respectively, expressed as the peak values of O 2 - production (mean ± SEM, n = 5). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05) and ns, not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Activity Assay, Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: C5a triggers a transient increase in the cytosolic concentration of free calcium ions ([Ca 2+ ] i ) in neutrophils, which is not affected by the allosteric FFA2R modulator Cmp58. Fura-2 loaded neutrophils were activated by propionate (25 µM) or different concentrations of C5a (2, 0.25, 0.1, and 0.05 nM) in the absence (upper panel) or presence (lower panel) of the allosteric FFA2R modulator Cmp58 (1 µM, pre-incubated with the cells for 10 min at 37 °C prior addition of the agonist). Results obtained are shown as one representative experiment out of 3. The arrows show the time point for the addition of the agonists to the Fura-2 labelled neutrophils.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Concentration Assay, Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: Activation of FFA2R inhibits (heterologously desensitizes) the neutrophil response induced by C5a. TNF-primed neutrophils incubated with Cmp58 (1 µM), were either left in a resting state (dashed lines) or activated by an FFA2R activating/transactivating ligand (solid lines) and the O 2 - production was measured continuously and expressed in Mcpm. When the response induced by the FFA2R activating agonist was terminated, the two cell samples were activated by an addition of C5a (2 nM). The results obtained in one representative experiment is shown together with an inset showing the peak activities induced by C5a (mean ± SEM, n = 3). (A) Propionate (25 µM), (B) ATP (25µM), and (C) AZ1729 (1µM) were used as the FFA2R activating ligand (the time point for addition is marked by arrow 1). The time point for addition of C5a to the cells is marked by arrow 2. Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05).

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Activation Assay, Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: C5a-induced desensitization in Cmp58-sensitized neutrophils. (A–C) TNF-primed neutrophils incubated with Cmp58 (1 µM, 5 min at 37 °C) were either left in a resting state (dashed lines) or activated by C5a (2 nM, solid line; the time point for addition is marked by arrow 1), and the O 2 - production was measured continuously and expressed as Mcpm. When the response, induced by the C5aR1 activating ligand was terminated, the two cell samples were activated by the addition of (A) propionate (25 µM, an FFA2R activating ligand), (B) ATP (50 µM, a P2Y 2 R activating ligand), or (C) AZ1729 (1 µM, an FFA2R activating ligand), and the time point for their addition is marked by arrow 2 (D–F) . The experimental setup differs from that described above, in that Cmp58 (1 µM), was added first when the response induced by C5a (time for addition marked by arrow 1), had settled. The neutrophils were then activated with either propionate [ (D) ; 25 µM)], ATP [ (E) ; 50 µM)], or AZ1729 [ (F) ; 1 µM)]. The time point for addition of the FFA2R activating/transactivating ligand is marked by arrow 2, and one representative experiment is depicted. The results obtained are shown together with an inset showing the peak activities induced by the FFA2R activating/transactivating ligands (mean ± SEM, n = 3). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as * ( p ≤ 0.05) and ns = not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: A proposed model for how the neutrophil NADPH oxidase is turned to an active state by the C5aR1 agonist C5a in the absence and presence of the allosteric FFA2R modulator Cmp58. (A) Left: The neutrophil response induced by a 2 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating NADPH oxidase and have also the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating oxidase and transactivate the allosterically modulated FFA2R; the transactivated FFA2R does not directly potentiate the NADPH oxidase response but reduces the inhibitory effect on this response, of the C5aR1 specific antagonist avacopan. (B) Left: The neutrophil response induced by a 0.1 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 have no NADPH oxidase activating effect. The signals have, however, the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 activate the O 2 -- generating NADPH oxidase, and this activation is totally dependent of the signals generated by the allosterically modulated FFA2R, a receptor made receptive to the transactivating signals generated by C5aR1.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Concentration Assay, Generated, Activation Assay

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

Journal: medRxiv

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

doi: 10.64898/2026.03.28.26349612

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

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

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

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-30853-1

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

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

Techniques: Marker, Expressing

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-30853-1

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

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

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-30853-1

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

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

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-30853-1

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

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

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-30853-1

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

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

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-025-30853-1

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

Techniques: Inhibition

(A) BMDM responses to (i) an initially homogeneous concentration of 10 nM C5a, (ii) negative control (no C5a), and (iii) an imposed gradient of 0–10 nM C5a. (B) Computational simulation, using parameters from experimental data, showing cells depleting C5a from a homogeneous 10 nM environment and migrating up the resulting self-generated gradient. (C) Self-generated gradient responses at (i) 10 nM C5a and (ii) 100 nM C5a. Red dashed box highlights the leading cell wave. Solid black and yellow lines indicate the distance covered by cells on the bridge at low and high concentrations, respectively.

Journal: bioRxiv

Article Title: Macrophage chemotaxis steered by complex self-generated gradients of complement C5a

doi: 10.1101/2025.11.05.686842

Figure Lengend Snippet: (A) BMDM responses to (i) an initially homogeneous concentration of 10 nM C5a, (ii) negative control (no C5a), and (iii) an imposed gradient of 0–10 nM C5a. (B) Computational simulation, using parameters from experimental data, showing cells depleting C5a from a homogeneous 10 nM environment and migrating up the resulting self-generated gradient. (C) Self-generated gradient responses at (i) 10 nM C5a and (ii) 100 nM C5a. Red dashed box highlights the leading cell wave. Solid black and yellow lines indicate the distance covered by cells on the bridge at low and high concentrations, respectively.

Article Snippet: Where appropriate, we used commercially available (Almac Sciences (Scotland) Ltd., Edinburgh) human C5a labelled with AlexaFluor-647 ® on its N-terminus.

Techniques: Concentration Assay, Negative Control, Generated

(A) Representative images showing BMDM migration after 6 h in initially homogeneous C5a concentrations of (i) 0.3 nM, (ii) 3 nM, and (iii) 30 nM. Red dashed boxes highlight the front wave of cells. (B) Negative control (no C5a), showing negligible migration. Red dashed line indicates the region assessed for cell migration. (C) Quantification of cells in the front wave at different C5a concentrations after 6 h. Data are shown as mean ± SD. Statistical significance was assessed using Student’s t -test; *** indicates p < 0.0001.

Journal: bioRxiv

Article Title: Macrophage chemotaxis steered by complex self-generated gradients of complement C5a

doi: 10.1101/2025.11.05.686842

Figure Lengend Snippet: (A) Representative images showing BMDM migration after 6 h in initially homogeneous C5a concentrations of (i) 0.3 nM, (ii) 3 nM, and (iii) 30 nM. Red dashed boxes highlight the front wave of cells. (B) Negative control (no C5a), showing negligible migration. Red dashed line indicates the region assessed for cell migration. (C) Quantification of cells in the front wave at different C5a concentrations after 6 h. Data are shown as mean ± SD. Statistical significance was assessed using Student’s t -test; *** indicates p < 0.0001.

Article Snippet: Where appropriate, we used commercially available (Almac Sciences (Scotland) Ltd., Edinburgh) human C5a labelled with AlexaFluor-647 ® on its N-terminus.

Techniques: Migration, Negative Control

(A) Distribution of fluorescent AF647-C5a after incubation without (i) or with (ii) BMDMs. (B) Quantification of fluorescence intensity along the bridge in the presence or absence of BMDMs, showing depletion of C5a near the cells. (C) Time course of C5a uptake. BMDMs were exposed to 5 nM AF647-C5a for 1 h, washed, and imaged at 0, 1.5, and 3 h. Fluorescence localized to intracellular puncta and accumulated near the perinuclear region, consistent with endocytosis and lysosomal processing. Images were captured and processed using identical acquisition parameters. (D) Magnified view showing AF647-C5a in vesicular structures 1.5h after exposure. Cells prepared as in C.

Journal: bioRxiv

Article Title: Macrophage chemotaxis steered by complex self-generated gradients of complement C5a

doi: 10.1101/2025.11.05.686842

Figure Lengend Snippet: (A) Distribution of fluorescent AF647-C5a after incubation without (i) or with (ii) BMDMs. (B) Quantification of fluorescence intensity along the bridge in the presence or absence of BMDMs, showing depletion of C5a near the cells. (C) Time course of C5a uptake. BMDMs were exposed to 5 nM AF647-C5a for 1 h, washed, and imaged at 0, 1.5, and 3 h. Fluorescence localized to intracellular puncta and accumulated near the perinuclear region, consistent with endocytosis and lysosomal processing. Images were captured and processed using identical acquisition parameters. (D) Magnified view showing AF647-C5a in vesicular structures 1.5h after exposure. Cells prepared as in C.

Article Snippet: Where appropriate, we used commercially available (Almac Sciences (Scotland) Ltd., Edinburgh) human C5a labelled with AlexaFluor-647 ® on its N-terminus.

Techniques: Incubation, Fluorescence

Different C5a inactivation mechanisms in mouse and human macrophages. A. A gradient of fluorescent C5a was established in an Insall chamber with & without mouse BMDM cells. B. measurement of fluorescent intensity along the bridge with and without BMDM cells, C. Imposed gradient of fluorescent C5a (i) without, & with THP-1 cells, & D. Measured intensity along the bridge with & without THP-1 cells, E. Dose response of chemotaxis. The number of cells that had migrated 200 μ m along the bridge was measured after 18hr for different C5a concentrations. G, H: Comparing the self-generated chemotaxis of mouse & human macrophages to 3nM C5a showed insignificant difference in their cosθ (G), but significant difference in the mean speed (H). Bar in G and H = SE (n=4). ** represents p-value < 0.001.

Journal: bioRxiv

Article Title: Macrophage chemotaxis steered by complex self-generated gradients of complement C5a

doi: 10.1101/2025.11.05.686842

Figure Lengend Snippet: Different C5a inactivation mechanisms in mouse and human macrophages. A. A gradient of fluorescent C5a was established in an Insall chamber with & without mouse BMDM cells. B. measurement of fluorescent intensity along the bridge with and without BMDM cells, C. Imposed gradient of fluorescent C5a (i) without, & with THP-1 cells, & D. Measured intensity along the bridge with & without THP-1 cells, E. Dose response of chemotaxis. The number of cells that had migrated 200 μ m along the bridge was measured after 18hr for different C5a concentrations. G, H: Comparing the self-generated chemotaxis of mouse & human macrophages to 3nM C5a showed insignificant difference in their cosθ (G), but significant difference in the mean speed (H). Bar in G and H = SE (n=4). ** represents p-value < 0.001.

Article Snippet: Where appropriate, we used commercially available (Almac Sciences (Scotland) Ltd., Edinburgh) human C5a labelled with AlexaFluor-647 ® on its N-terminus.

Techniques: Chemotaxis Assay, Generated

Receptor mediated C5a uptake via C5aR1. Time-lapse images of cells subjected to A. 0-10nM C5a with cells tracks, B. 0-10nM C5a in the presence of 10uM PMX-53 (inhibitor), C. Spider plot for the 0- 10nM C5a, D. Spider plot for the 0-10nM C5a in the presence of 10uM PMX-53, E. Comparison of chemotaxis index for both the cases with and without inhibitor, F. Confocal images indicating the C5a AF647® uptake and G. quantification of cellular intensity (bars show mean± SEM, n=3; ** student’s t-test p-value <0.001)

Journal: bioRxiv

Article Title: Macrophage chemotaxis steered by complex self-generated gradients of complement C5a

doi: 10.1101/2025.11.05.686842

Figure Lengend Snippet: Receptor mediated C5a uptake via C5aR1. Time-lapse images of cells subjected to A. 0-10nM C5a with cells tracks, B. 0-10nM C5a in the presence of 10uM PMX-53 (inhibitor), C. Spider plot for the 0- 10nM C5a, D. Spider plot for the 0-10nM C5a in the presence of 10uM PMX-53, E. Comparison of chemotaxis index for both the cases with and without inhibitor, F. Confocal images indicating the C5a AF647® uptake and G. quantification of cellular intensity (bars show mean± SEM, n=3; ** student’s t-test p-value <0.001)

Article Snippet: Where appropriate, we used commercially available (Almac Sciences (Scotland) Ltd., Edinburgh) human C5a labelled with AlexaFluor-647 ® on its N-terminus.

Techniques: Comparison, Chemotaxis Assay

Review

recombinant human c5a rhc5a (MedChemExpress)

Structured Review

Images

1) Product Images from "The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan"

Related Articles

Isolation:

Incubation:

Similar Products

Image Search Results