Mouse igg1 fab and f ab 2 preparation kit

Histopaque1077 for the purification of PBMCs was obtained from Sigma-Aldrich (St. Louis, MO). PE-labeled mouse anti-human CD14 (clone M5E2) was purchased from BD Biosciences (San Jose, CA). Allophycocyanin-labeled mouse anti-human CD16 (3G8), mouse anti-human CD41 (HIP8), mouse anti-human CD42b (HIP1), mouse anti-human CD9 (HI9a), and mouse anti-human CD36 (5-271) were obtained from BioLegend (San Diego, CA). Mouse anti-human CD32 (IV.3) was obtained from StemCell Technologies (Vancouver, Canada). F(ab’)₂ of the anti-human CD41 and isotype control mAbs were prepared using a Mouse IgG1 Fab and F(ab’)₂ Preparation Kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. Other murine mAbs as to human platelet antigens including #33 anti-human CD61 IgG2a were generated by Medical & Biological Laboratories, Co., Ltd. (Nagoya, Japan). The characters of several mAbs are summarized in Additional file 1: Table S1.

38–2 Fab and control mouse IgG Fab fragments were prepared using a Mouse IgG₁ Fab and F(ab’)₂ Preparation Kit (Thermo Scientific Inc., Waltham, MA). In brief, 38–2 mAb and control mouse IgG (Sigma) were respectively subjected to proteolysis with immobilized ficin. The digested Fc portion and undigested IgG fragment were removed by passing over a column of immobilized protein A 3 times and the resulting elutes containing the Fab fragments was collected. The Fab concentration was determined at 280 nm using a Nanodrop 1000 spectrophotometer (Thermo Scientific Inc.).