Limulus amebocyte lysate assay

LAMPs were prepared as described previously (He et al., 2009 (link)). Briefly, M. hyopneumoniae was cultivated in a mycoplasma medium until the beginning of the stationary growth phase (when a red pH indicator turned orange) and then collected by centrifugation. M. hyopneumoniae cells were washed with PBS twice and resuspended in 5 ml of Tris‐buffered saline (TBS; 50 mM Tris‐Cl, pH 8.0, 0.15 M NaCl) containing 1 mM EDTA (TBSE), to which Triton X‐114 was added to a final concentration of 2% and incubated at 4°C for 1 h. The lysate was then incubated at 37°C for 10 min for phase separation. After centrifugation, the upper aqueous phase was removed and replaced with the same volume of TBSE. The solution was vortexed and incubated at 4°C for 10 min. The phase separation process was repeated twice. The final Triton X‐114 phase was resuspended in TBSE to the original volume, and 2.5‐fold volumes of ethanol were then added to precipitate the membrane components overnight at −20°C. After centrifugation, the pellet was resuspended in PBS and lysed by sonication. Protein concentrations were examined using the Bradford assay (Thermo Scientific, Waltham, MA, USA). The endotoxin concentration of the heat‐inactivated mycoplasma LAMPs was <0.04 endotoxin units/ml, as checked by the Limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA, USA).

The polyclonal anti-β2GPI antibodies were purified from sera of New Zealand rabbits immunized with human β2GPI peptide sequence (35GYVSRGGMRKFICPLTG51) according to our previous methods (23 (link)). The purified anti-β2GPI IgG could recognize human and mouse β2GPI as demonstrated by western blotting and ELISA (23 (link)). Our previous study demonstrated that this anti-β2GPI IgG could induce an APS mouse model. The isotype control antibodies (NR-IgG) from sera of normal rabbits were purified by Protein G Sepharose columns (GE Healthcare, Chicago, IL, USA). All the IgG samples and reagents were subjected to Detoxi-Gel™ (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to remove endotoxin contamination (<0.03 EU/ml) using the Limulus amebocyte lysate assay (Associates of Cape Cod, Inc., Falmouth, MA, USA).
C3H/HeN and C3H/HeJ mice (n=8 per treatment group) were twice injected intraperitoneally with anti-β2GPI (100 µg) or NR-IgG (100 µg) at 0 and 48 h. Surgical procedures to obtain peritoneal macrophages or aortas were performed at 72 h after the first injection. In addition, other groups of C3H/HeN and C3H/HeJ mice (n=8 in each group) were challenged with LPS (1 µg/g body weight; E. coli serotype O111:B4; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) 2 h before surgical procedures as the positive control.