recombinant mouse c5a protein  (R&D Systems)

Journal: bioRxiv

Article Title: SARM1 is required for macrophage immunophenotype switching that is essential for nerve repair

doi: 10.64898/2026.04.07.716973

Figure Lengend Snippet: ( A ) Representative still images from live cell imaging sequences following a 2D scratch assay of WT and Sarm1-/- stimulated Mɸ. t=0 is immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( B,C ) Quantification of the relative wound density of WT ( B ) or Sarm1-/- ( C ) Mɸ cultures after the scratch assay in A represented as a percentage of t=0. N=3 biological replicates. Error = SEM. *p<0.05; **p<0.01; ***p<0.005; letters (a,b,c) p<0.05. by two-way repeated measures ANOVA with Dunnett post-hoc test. Blue asterisk = mCSF compared to t=0. Green asterisk = IL-4 compared to t=0. Orange asterisk = LPS compared to t=0. a = mCSF compared to IL-4; b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( D ) Representative still images from live cell imaging sequences following a 3D invasion assay in Matrigel of WT and Sarm1-/- stimulated Mɸ. t=0 immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( E ) Quantification of the relative wound density of the invasion assay without C5a added (top) and with C5a added as a chemoattractant (bottom) represented as a percentage of t=0. N=2-4 biological replicates. Error = SEM. Letters (b,c) p<0.05; **p<0.01; ***p<0.005; ****p<0.001 by two-way ANOVA, main effects model with Tukey post-hoc test after simple linear regression model indicated slopes were not different. Wildtype Mɸ + C5a main effects could not be calculated because slopes were significantly different. Orange asterisk = LPS compared to t=0. b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( F ) Representative images of F4/80 (green) positive Mɸ stained for Oil Red O lipids (magenta) following a myelin challenge assay. No clearance indicates a 24 hour myelin treatment and clearance indicates 24 hours of myelin treatment followed by myelin removal for 24 hours. Scale bar = 100µm. ( G ) Quantification of OilRedO intensity per Mɸ (normalized to Mɸ cell area). N = 3 biological replicates and each data point = 1 Mɸ. Mean +/- SEM is shown. Outliers were removed with ROUT Q=1. **p<0.01; ***p<0.005; ****p<0.001 by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Black asterisk = WT to Sarm1-/- comparisons. Purple asterisk = Sarm1-/- to Sarm1-/- comparisons. Blue asterisk = WT to WT comparisons.

Article Snippet: Additional media with or without recombinant mouse complement component C5a (R&D system, #2150-C5/CF) was added after incubation.

Techniques: Live Cell Imaging, Wound Healing Assay, Invasion Assay, Staining

Journal: Allergy

Article Title: Role for Complement C5 in Eosinophilic Inflammation of Severe Asthma.

doi: 10.1111/all.16616

Figure Lengend Snippet: FIGURE 6 | Complement C5 alone promotes eosinophil recruitment and activation in IL-5 transgenic mouse. (a) Experimental scheme of daily challenge of an IL-5 transgenic mouse (IL5-tg) with recombinant mouse C5. (b) Change in baseline airway resistance during acetylcholine airway challenge in IL5-tg mice treated with either phosphate-buffered saline (PBS) or Complement C5. (c) Bronchoalveolar lavage fluid (BALF) eosinophil percentage (CD45+live+CD11c−SiglecF+). (d) BALF eosinophil cell count (e, f) BALF eosinophil granule proteins of mouse eosinophil peroxidase (EPX) and mouse eosinophil cationic protein ECP (RNASE3) levels in IL5-tg mice treated with phosphate-buffered saline (PBS) or Complement C5. (g) Peribronchial inflammatory infiltration and inflammatory score, (h) Periodic Acid-Schiff (PAS) staining for epithelial goblet cell metaplasia and PAS positive cells per bronchi. Data show mean ± SD. Data was analyzed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. **p < 0.01. N = 5 each group, all experiments were performed by different operators and repeated twice. The data presented in the main figures represent the results from one batch of experiments, which consistently showed similar trends across the repetitions.

Article Snippet: For the C5 stimulating IL- 5 transgenic mice model, 500 ng recombinant C5 (R&D SYSTEMS; Cat# 2150- C5) alone in 40 μL of sterile PBS or vehicle PBS was administrated via nasal instillation every 2 days for 2 weeks.

Techniques: Activation Assay, Transgenic Assay, Recombinant, Saline, Cell Counting, Staining

Journal: Allergy

Article Title: Role for Complement C5 in Eosinophilic Inflammation of Severe Asthma.

doi: 10.1111/all.16616

Figure Lengend Snippet: FIGURE 2 | Complement C3 and C5 expression in sputum and association of C5 with eosinophils. Box plot of (a) C3 and (b) C5 expression in sputum between control and severe asthma. Box plot of (c) C3 and (d) C5 expression profiles among control, high-eosinophil (Eoshigh-SA) and low- eosinophil (Eoslow-SA) severe asthma. (e) Correlation of C5 and eosinophilic inflammatory indicators in severe asthma including sputum eosinophil percentage (Sputum_Eos(%)), fractional level of nitric oxide in exhaled breath (FENO), blood eosinophil count (Blood_Eos (×109/L)) and blood eosin- ophil percentage (Blood_Eos(%)). Box plots show the median and interquartile range, with whiskers representing the maximum and minimum val- ues. Eoshigh-SA was defined as sputum eosinophil percentage ≥ 3%, Eoslow-SA < 3%. Rho represents Spearman rank correlation coefficient. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Eoshigh-SA, eosinophil-high severe asthma; Eoslow-SA, eosinophil-low severe asthma; SA, severe asthma.

Article Snippet: For the house dust mite (HDM) and lipopolysaccharide (LPS)- induced steroid- resistance airway allergic model [9], 50 μg of HDM (XPB91D3A25; Greer Laboratories) with 500 ng of LPS (L4391, Escherichia coli 0111:B4, Sigma- Aldrich) dissolved in 40 μL of sterile phosphate- buffered saline (PBS) was introduced into airways once daily for three continuous days (days 0–2) during allergen sensitisation; 500 ng recombinant complement C5 (R&D SYSTEMS; Cat#2150- C5) or 500 μg anti- C5 neutralization antibody (HycultBiotech; Cat# HM1073) was challenged i.n during days 15–19 continuously.

Techniques: Expressing, Control

Journal: Allergy

Article Title: Role for Complement C5 in Eosinophilic Inflammation of Severe Asthma.

doi: 10.1111/all.16616

Figure Lengend Snippet: FIGURE 3 | Correlation of the change of C5 and of related variables between baseline and longitudinal visit. Pearson correlation of the change of (a) FEV1 (%Predicted), (b) Eosinophil -Derived Neurotoxin (RNASE2), (c) Eosinophil Cationic Proteins (RNASE3), (d) Eosinophil Granule Major Basic Protein (PRG2), (e) Eosinophil Peroxidase (EPX), (f) Sputum eosinophil percentage, (g) Blood eosinophil percentage, (h) Blood eosinophil counts with the change in expression of complement C5. Correlation coefficient rho and p values are shown. p < 0.05 is statistically significant.

Article Snippet: For the house dust mite (HDM) and lipopolysaccharide (LPS)- induced steroid- resistance airway allergic model [9], 50 μg of HDM (XPB91D3A25; Greer Laboratories) with 500 ng of LPS (L4391, Escherichia coli 0111:B4, Sigma- Aldrich) dissolved in 40 μL of sterile phosphate- buffered saline (PBS) was introduced into airways once daily for three continuous days (days 0–2) during allergen sensitisation; 500 ng recombinant complement C5 (R&D SYSTEMS; Cat#2150- C5) or 500 μg anti- C5 neutralization antibody (HycultBiotech; Cat# HM1073) was challenged i.n during days 15–19 continuously.

Techniques: Derivative Assay, Expressing

Journal: Allergy

Article Title: Role for Complement C5 in Eosinophilic Inflammation of Severe Asthma.

doi: 10.1111/all.16616

Figure Lengend Snippet: FIGURE 6 | Complement C5 alone promotes eosinophil recruitment and activation in IL-5 transgenic mouse. (a) Experimental scheme of daily challenge of an IL-5 transgenic mouse (IL5-tg) with recombinant mouse C5. (b) Change in baseline airway resistance during acetylcholine airway challenge in IL5-tg mice treated with either phosphate-buffered saline (PBS) or Complement C5. (c) Bronchoalveolar lavage fluid (BALF) eosinophil percentage (CD45+live+CD11c−SiglecF+). (d) BALF eosinophil cell count (e, f) BALF eosinophil granule proteins of mouse eosinophil peroxidase (EPX) and mouse eosinophil cationic protein ECP (RNASE3) levels in IL5-tg mice treated with phosphate-buffered saline (PBS) or Complement C5. (g) Peribronchial inflammatory infiltration and inflammatory score, (h) Periodic Acid-Schiff (PAS) staining for epithelial goblet cell metaplasia and PAS positive cells per bronchi. Data show mean ± SD. Data was analyzed with one-way analysis of variance (ANOVA) with Tukey's post hoc test. **p < 0.01. N = 5 each group, all experiments were performed by different operators and repeated twice. The data presented in the main figures represent the results from one batch of experiments, which consistently showed similar trends across the repetitions.

Article Snippet: For the house dust mite (HDM) and lipopolysaccharide (LPS)- induced steroid- resistance airway allergic model [9], 50 μg of HDM (XPB91D3A25; Greer Laboratories) with 500 ng of LPS (L4391, Escherichia coli 0111:B4, Sigma- Aldrich) dissolved in 40 μL of sterile phosphate- buffered saline (PBS) was introduced into airways once daily for three continuous days (days 0–2) during allergen sensitisation; 500 ng recombinant complement C5 (R&D SYSTEMS; Cat#2150- C5) or 500 μg anti- C5 neutralization antibody (HycultBiotech; Cat# HM1073) was challenged i.n during days 15–19 continuously.

Techniques: Activation Assay, Transgenic Assay, Recombinant, Saline, Cell Counting, Staining

Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Library size-normalized counts detected by RNA sequencing in mouse RPM and in M0-differentiated BMDM (3 mice each; only GPCRs with average count >15 displayed). B Gprc5b expression in RPM and lymph node-derived lymphocytes was determined by NanoString RNA analysis (cells pooled from two mice per data point). C Knockout efficiency in RPM from control mice (white) and M-G5b-KOs (gray) was analyzed by qRT-PCR (C, n = 4, data normalized to Gapdh and controls set to 1). D Knockout efficiency in RPM was analyzed by immunoblotting (unspecific band around 38 kDa; the higher of the two specific bands probably represents glycosylated GPRC5B ; GAPDH as loading control). E, F Expression of Nos2 (E) and Tnf (F) was determined in RPM by qRT-PCR under basal conditions and after 6 h of stimulation with 1 µg/ml LPS ( n = 11/12/12/12 in E, 12/10/11/12 in F), data normalized to Gapdh and control set to 1). G Basal and C5a (20 ng/ml)-induced transwell migration in RPM (all cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration) ( n = 3). H The distance travelled by individual RPM in response to different chemotactic factors (C5a, 20 nM; CCL5, 10 ng/ml; fMLP, 10 nM) was determined by live cell imaging ( n = 512 cells from 2 mice per group; cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration, arb. units: arbitrary units). Phagocytic activity of LPS (1 µg/ml, 6 h)-stimulated RPM was determined by uptake of pHrodo E. coli bioparticles ( I , J , n = 5) or pHrodo-labeled apoptotic thymocytes ( K , L n = 10); I + K show original traces, J + L statistical evaluation of areas under the curve (AUC). Body weight change ( M ) and bacterial colony-forming units in peritoneal lavage fluid harvested 24 h after injection of fecal bacteria ( N ) ( n = 10). O, P Numbers of CD11b + , F4/80 + , MHCII - , Tim4 + RPM and CD11b + , F4/80 lo , MHCII + , CCR2 + BMDM before and 3, 24, and 54 h after i.p. injection of fecal bacteria ( n = 3/3/3/3/11/12/5/5). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t -test (C, J, L, N), two-way ANOVA (E-H) or two-way RM-ANOVA (M) with Sidak’s multiple comparison test, unpaired two-sided t-test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (O, P). * P < 0.05; *** P < 0.001; **** P < 0.0001; n , number of individual mice. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: RNA Sequencing, Expressing, Derivative Assay, Knock-Out, Control, Quantitative RT-PCR, Western Blot, Migration, Live Cell Imaging, Activity Assay, Labeling, Injection, Bacteria, Comparison

Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Knockout efficiency was determined by qRT-PCR in RPM and M0 BMDM (data normalized to Gapdh and RPM controls set to 1) ( n = 12). Analyses in resting and LPS (1 μg/ml, 6 h)-stimulated M0 BMDM: Expression of inflammatory genes ( B , C ; n = 15/14/15/15 in B, 15/14/15/15 in C), production of NOx ( D ; n = 3) or release of cytokines ( E , n = 3). F Transwell migration of M1 BMDM in response to different chemotactic factors ( n = 6) (CCL5: 75 ng/ml, CCL2: 10 ng/ml, SDF-1β: 100 ng/ml, C5a: 20 ng/ml, fMLP: 10 nM). Uptake of pHrodo E.coli fragments by M0 BMDM: G , exemplary curves; H , statistical analysis of AUC ( n = 6). I Flow cytometric analysis of CD11b-positive cells in the combined infarct and border zones of hearts harvested 4 days after infarction ( n = 5). J Echocardiographic analysis of ejection fraction (EF%) before and after infarction (8 controls, 4 KOs). K Histological analysis of scar size in hearts harvested 21 days after infarction ( n = 7 controls, 4 KOs), left ventricle (LV). L, M DSS colitis: Disease activity index integrating body weight change, stool consistency, intestinal bleeding (L) and colon length on day 6 (M) ( n = 7(L), 7/7/10/11 in M). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t- test (A, H, K), two-way ANOVA with Sidak’s multiple comparisons test (B-F, J, M), unpaired two-sided t -test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (I), two-way repeated measures ANOVA with Sidak’s multiple comparisons test (L). n, number of mice per group; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Migration, Activity Assay