Glass coverslips #1.5 were coated with neutravidin (Thermo scientific) at 0.5 mg/mL in PBS, at 4°C for 4 hours. After washes, coverslips were incubated with 10 μg/mL biotin-conjugated M1/70 (BD Pharmingen 553309) or rat IgG2b isotype control (BD Pharmingen 553987) antibodies at 4°C overnight. Coverslips were then blocked with PLL-PEG (Susos) at 0.1 mg/mL, at 4°C for at least 2 hours. Macrophages were suspended in ice cold PBS with 2 mM EDTA by scraping, then washed in serum free RMPI 1640, supplemented with 25 mM HEPES pH 7.3 and 150 ng/ml PMA 15 minutes before addition onto the coverslips. Cells were imaged immediately for live cell microscopy, or fixed with 4% PFA after 5 minutes and processed for immunofluorescence.
For frustrated phagocytosis on polyacrylamide gels and traction force microscopy, 40 μm thick gels with 40 nm TransFluoSpheres (633/720) (Thermo scientific) were prepared on glass coverslips as described before35 (link). neutravidin (0.5 mg/mL) was crosslinked to the surface of the gels using Sulfo-SANPAH at 1mg/ml (Thermo scientific). After washes, gels were incubated with biotin-conjugated M1/70 (BD Pharmingen 553309) or rat IgG2b isotype control (BD Pharmingen 553987) antibodies at 10 μg/mL, at 4°C overnight.
For frustrated phagocytosis on polyacrylamide gels and traction force microscopy, 40 μm thick gels with 40 nm TransFluoSpheres (633/720) (Thermo scientific) were prepared on glass coverslips as described before35 (link). neutravidin (0.5 mg/mL) was crosslinked to the surface of the gels using Sulfo-SANPAH at 1mg/ml (Thermo scientific). After washes, gels were incubated with biotin-conjugated M1/70 (BD Pharmingen 553309) or rat IgG2b isotype control (BD Pharmingen 553987) antibodies at 10 μg/mL, at 4°C overnight.