Anti c4d monoclonal antibody

Primary antibodies were anti-C4d monoclonal antibody (Quidel, San Diego, CA) (murine anti-human monoclonal C4d, dilution 1:250), anti-C3d monoclonal antibody (Quidel, San Diego, CA) (murine anti-human monoclonal C3d, dilution 1:25), anti-C5b-9 polyclonal antibody (Abcam, Cambridge, MA) (rabbit polyclonal to C5b-9, dilution 1:100) and IgG1k-isotype control (BioLegend, San Diego, CA). Secondary antibody was Alexa Fluor 488-conjugated rabbit anti-mouse IgG and donkey anti-rabbit IgG (Life technologies, Grand Island, NY) (dilution 1:100). Other reagents were as follows: Hanks’ balanced salt solution with Ca²⁺ and Mg²⁺ (HBSS⁺⁺) (Life technologies), IgG-free bovine serum albumin (BSA) (Sigma-Aldrich, St Louis, MO), DAF-FM diacetate (Life technologies) and eosin-5-maleimide (Life technologies).

Primary antibodies were anti-C4d monoclonal antibody (A213; Quidel, San Diego, CA) and IgG1k-isotype control (401402; BioLegend, San Diego, CA). Secondary antibody was Alexa Fluor 488-conjugated rabbit anti-mouse IgG (A11059; Life technologies, Grand Island, NY). Other reagents were as follows: Hanks’ balanced salt solution with Ca²⁺ and Mg²⁺ (HBSS⁺⁺) (14025; Life technologies), IgG-free bovine serum albumin (BSA) (A9085; Sigma-Aldrich, St Louis, MO), DAF-FM diacetate (D23844; Life technologies), Flio-4 AM (F14217; Life technologies), and eosin-5-maleomide (E118; Life technologies).

Recruitment of C4BP by NS1 to the cell surface followed by measurement of the cofactor activity of C4BP was performed according to a previous study (25 (link)). Briefly, serum-free supernatants (200 μl) containing 12 μg/ml of NS1 from DENV-infected C6/36, DENV-infected PscloneD cells or S2-NS1 cells were incubated with C4BP (2.5 μg/ml, 50 μl) for 1 h on ice. Adherent BHK cells (1 × 10⁶) were detached by PBS supplemented with 8 mM EDTA. Subsequently, 10% FBS was added to the C4BP solution (with or without NS1) and incubated for 1 h on ice. After three washes with 1 ml DMEM at 4°C, cells were washed with 1 ml dextrose veronal buffered saline (DVB, 2.5 mM veronal buffer pH 7.35, 25 mM NaCl, and 240 mM glucose) at 4°C. C4b (400 ng) and factor I (200 ng) diluted in DVB were added to cell pellets in a total volume of 50 μl. After a 15-min incubation at 37°C, cells were pelleted and supernatants were mixed with SDS sample buffer supplemented with 2-mercaptoethanol (5% v/v) and subjected to 12% SDS-PAGE. C4b fragmentation was analyzed by Western blotting using a 1:2,000 dilution of anti-C4d monoclonal antibody (Quidel) followed by a 1:5,000 dilution of HRP-conjugated anti-mouse IgG.