Control igg from mouse serum

Ca9-22 cells derived from human gingival squamous cell carcinoma were provided from the Japanese Collection of Research Bioresources (JCRB) (Osaka, Japan) and cultured in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL) (Nacalai Tesque) and streptomycin (100 mg) (Nacalai Tesque) at 37°C in a humidified atmosphere with 5% CO₂. BLM was purchased from LKT Laboratories (St. Paul, MN, USA). Anti-EGFR antibody was prepared as described previously [13 (link)]. Briefly, culture supernatants from the 528 hybridomas (ATCC; TKG 0555, Manassas, VA, USA) were collected and fractionated with 60% ammonium sulfate to prepare the anti-EGFR antibody, and the final pellet, which contained the crude anti-EGFR antibody, was dissolved in phosphate-buffered saline (PBS). The crude anti-EGFR antibody was then purified using a Nab Protein A plus Spin Kit (PIERCE, Rockford, IL, USA). Control IgG from mouse serum was purchased from SIGMA ALDRICH (St. Louis, MO, USA).

MV-TF activity and MV-FXa generation was measured with an adapted method from Wang et al [24 (link)]. First, 600 μL plasma was diluted in 1 mL HBSA buffer (137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose, 10 mM HEPES, 0.1% (w/v) bovine serum albumin, pH 7.4) and centrifuged at 20,000 × g for 15 minutes at 4°C in order to pellet microvesicles. The pellets were washed once in 1 mL HBSA and resuspended in 180 μL HBSA. The samples were then incubated with monoclonal mouse anti-CD142 antibody (clone HTF-1, BD Pharmingen) or control IgG from mouse serum (Sigma-Aldrich) for 15 minutes at room temperature in a 96-well plate. After incubation, 50 μL HBSA containing 10 mM CaCl_2, 73 nM FX (Enzyme Research Laboratories, South Bend, IN, USA), and 2.4 nM factor VIIa (Enzyme Research Laboratories) was added to each sample and incubated for two hours at 37°C. The reaction was stopped by addition of 25 μL HBSA containing 25 mM EDTA. Then, 25 μL of 4 mM chromogenic Pefachrome FXa 8595 (Pentapharm, Basel, Switzerland) was added to the wells and incubated at 37°C for 15 minutes. The plate was read at absorbance 405 nm on a Fluostar Optima (BMG Labtech, Ortenberg, Germany). Innovin (Siemens Healthcare, Erlangen, Germany) was used to generate a standard curve to calculate the procoagulant activity of microvesicles.