Mouse plasma

The interaction of Fc variants with mouse C1q was measured by ELISA. MaxiSorp 384-well plates (Thermo Fisher Scientific) were directly coated with the anti-CD154 antibodies overnight at 4 °C. After blocking with TBS-T containing 0.5% BSA and 1 × Block Ace (DS Pharma Biomedical) for 7 h at 4 °C, 10% mouse plasma (Innovative Research, Novi, MI, USA) was added onto the plates and incubated overnight at 4 °C. At room temperature, the plates were incubated with a biotinylated anti-mouse C1q antibody (Hycult Biotech, Uden, The Netherlands) for 1 h. Pierce High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific) was added to react for 1 h at room temperature. Washes in PBS-T (pH 7.4) were performed after each subsequent step. ABTS peroxidase substrate (SeraCare Life Sciences, Milford, MA, USA) was subsequently added, and the signal was measured by a plate reader at a wavelength of 405 nm.

Commercially available RE was purchased from Vitiva (Markovci, Slovenia). CA and CL were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Acetonitrile and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Butyl paraben was purchased from Acros Organics (Fair Lawn, NJ, USA). Mouse plasma was purchased from Innovative Research Inc. (Novi, MI, USA). Dextran sodium sulfate was purchased from Alfa Aesar (Haverhill, MA, USA). RNAlater was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fluorescein isothiocyanate (FITC)–dextran (40 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sestrin 2, zonula occludens-1 (ZO-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit polyclonal primary antibodies were purchased from Proteintech (Rosemont, IL, USA). Horseradish peroxidase (HRP)-linked anti-rabbit secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).