Pierce mouse igg1 fab and f ab 2 micro preparation kit

SN3 F(ab’)₂ was prepared using the Pierce™ Mouse IgG1 Fab and F(ab’)₂ Micro Preparation Kit (Thermo Fisher Scientific). F(ab’)₂ concentration was measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific), and size was confirmed using non-reducing SDS-PAGE and Western blot. Alternatively to F(ab’)₂ fragments, the Fc-domain of full anti-CD24-mIgG1 (Clone SN3) was blocked with goat F(ab’)₂ anti-mouse IgG1 (Fc-domain specific). For that, the full antibody was incubated with goat F(ab’)₂ in a 1:5 molar ratio for 30 min at RT. A volume of the mix corresponding to a final concentration of 1 μg/mL of full antibody was used in phagocytosis assays.

CD47 F(ab’)2 fragments were produced from ficin digest of IgG using Pierce Mouse IgG1 Fab and F(ab’)2 Micro Preparation Kit (Thermo Fisher Scientific), according to the manufacture’s recommendations. The purified anti-CD47 F(ab’)2 fragments were collected for the efferocytosis assay.