recombinant mouse c3a  (PeproTech)

Journal: Cells

Article Title: House Dust Mite Nebulization Drives Alarmin and Complement Activation in a Murine Tracheal Air–Liquid Interface Culture System

doi: 10.3390/cells14201598

Figure Lengend Snippet: Time-dependent complement activation and anaphylatoxin receptor expression in ALI cultures exposed to HDM. ( a ) C3 and C3a concentrations in cell lysates and the SN of unstimulated (steady-state) ALI cultures and 24, 48 and 72 h after HDM exposure (steady-state, 24, 48 and 72 h: n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001. ( b ) HDM and HI-HDM mediated cleavage of hC3 into hC3a ( n = 3 independent experiments). Data are shown as mean ± SEM. Data were analyzed using an unpaired T -test. *** p < 0.001. ( c ) C5 and C5a concentrations in cell lysates and the SN of steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). ( d ) Comparison of C3 and C5 ( left panel ), C3a and C5a ( right panel ) concentrations in cell lysates from steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. **** p < 0.0001. ( e ) Immunofluorescence analysis of C3aR and C5aR1 expression in unstimulated ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM ( n = 3). Data were analyzed using one-way ANOVA with Holm–Šidák’s multiple comparisons test. * p < 0.05; ** p < 0.01. The number of experiments shown in ( a , c – e ) refers to biological replicates derived from 6 independent preparations of trachea, each representing a separate cell isolation and culture.

Article Snippet: Plates were washed four times with 0.05% Tween 20 in PBS; serial dilutions of recombinant mouse C3a (R&D Systems #8085-C3-025—Minneapolis, MN, USA) were performed; and samples were added (25 μL/well) and incubated at RT for 90 min. After washing four times with 0.05% Tween 20 in PBS, 25 μL/well of 1 μg/mL in 1% BSA/PBS biotinylated anti-mouse C3a detection antibody (clone I87-419, BD Biosciences #558251—San Jose, CA, USA) was added and incubated for 1 h at RT.

Techniques: Activation Assay, Expressing, Comparison, Immunofluorescence, Derivative Assay, Cell Isolation

Journal: Journal of Neuroinflammation

Article Title: Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging

doi: 10.1186/s12974-024-03130-7

Figure Lengend Snippet: Microglial C3a – C3aR signaling promotes glycolysis and phagocytosis. Young microglia (postnatal day 0–2) were treated with 10 nM recombinant mouse C3a. ( a - d ) p-AKT (Ser473), p-mTOR (Ser2448) and HIF1α were assessed by Western blotting at the indicated timepoints (0–24 h), and expression was normalized to total AKT, total mTOR and β-actin, respectively. n = 5 replicates. Data are presented as mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA). ( e - g ) Seahorse assays were used to evaluate real-time glycolytic rate in young microglia (postnatal day 0–2, pooled male and female) after 18 h in vitro treatment with 10 nM recombinant mouse C3a. Stimuli were added as indicated ( e ), and basal glycolysis ( f ) and compensatory glycolysis ( g ) were determined by calculating the glycolytic Proton Efflux Rate (e; glycoPER). n = 5/group. # p < 0.05 (unpaired t-test). ( h , i ) Flow cytometry analysis was performed to assess phagocytosis of FITC-fAβ 1−42 . Rapamycin (50 µM) or 2-DG (5 mM) were pre- (1 h) and co-treated (18 h) with C3a. Proportion of phagocytic cells ( h ) was assessed by evaluating FITC-positive microglia, and FITC MFI was evaluated in total live microglia ( i ). Data are presented as mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA)

Article Snippet: Primary microglia were stimulated with vehicle (PBS) or recombinant mouse C3a (10 nM; Peprotech) for the indicated time as in each figure legend.

Techniques: Recombinant, Western Blot, Expressing, In Vitro, Flow Cytometry