mouse anti c5  (Proteintech)

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: High-binding 96-well plates (Corning #9018—Corning, NY, USA) were coated overnight at 4 °C with 50 μL/well of 2 μg/mL anti-mouse C5 capture antibody (clone BB5.1, Hycult Biotech #HM1073—Uden, Netherlands).

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

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: C3aR, C5 and C5aR1 expression in ALI cultures before and after HDM stimulation. ( a – d ) C5aR1 expression (red) in ( a ) unstimulated ALI culture and ( b ) 24 h, ( c ) 48 h or ( d ) 72 h after HDM exposure. ( e – h ) C3aR expression (red) in ( e ) unstimulated ALI culture and ( f ) 24 h, ( g ) 48 h or ( h ) 72 h after HDM exposure. ( i ) Z-stack 3D reconstruction of ALI culture showcasing the heterogeneity of AE cells in unstimulated ALI culture and the patchy expression of C5 and C5aR1. Ciliated cells (purple: acetylated tubulin), goblet cells (green: MUC5AC), C5 (red), C5aR1 (yellow); white circles point toward overlap of C5 and C5aR1 expression (orange). Nuclear stain is in gray (Hoechst 33342). Red arrows point toward C5 staining; yellow arrows indicate C5aR1 staining; and green arrows point toward goblet cells. The two red lines seen horizontally are part of the “view box” from the Leica microscope software (LAS X 4.8.1.29271—Wetzlar, Germany). Scale bar = 50 µm.

Article Snippet: High-binding 96-well plates (Corning #9018—Corning, NY, USA) were coated overnight at 4 °C with 50 μL/well of 2 μg/mL anti-mouse C5 capture antibody (clone BB5.1, Hycult Biotech #HM1073—Uden, Netherlands).

Techniques: Expressing, Staining, Microscopy, Software