Nucblue live ready probes hoechst 33342 dye reagent

To quantitate phagocytosis following transfection of 1 × 10⁵ RAW 264.7 cells with mimics and inhibitors, pHrodo deep red E. coli bioparticles conjugate for phagocytosis (Invitrogen, P35360) were used according to the manufacturer’s protocol. Briefly, following incubation in a 96-well black polystyrene clear-bottom tissue culture–treated microplate (Corning), the culture medium was removed, and bioparticles prepared in live cell imaging solution (Invitrogen) were added to the wells. Following incubation of 45 min at 37°C, fluorescence was measured at emission excitation of 640/680 and represented as total radiant efficiency. For imaging, the bioparticles were removed and washed with PBS. Cells were stained with NucBlue live-ready probes (Hoechst 33342 dye) reagent (Invitrogen). The stain was removed after 10 min of incubation and washed with PBS. live cell imaging solution was added to the cells and imaged using Evos FL Auto 2 (Invitrogen).

To quantitate Fcgr-mediated phagocytosis, following transfection of 1 × 10⁵ RAW 264.7 cells with mimics and inhibitors, Cayman’s IgG (fluorescein isothiocyanate (FITC)-conjugated) phagocytosis kit (Cayman, 500290) was used according to the manufacturer protocol. Briefly, following incubation in a 96-well black polystyrene clear-bottom tissue culture–treated microplate (Corning), the culture medium was removed, and IgG particles prepared in DMEM were added to the wells. Following incubation of 1 h at 37°C, the medium with IgG was removed, cells were washed with PBS, and cell surface–bound fluorescent particles were quenched by trypan blue. After PBS wash, fluorescence was measured at emission excitation of 465/540 and represented as total radiant efficiency. For imaging, IgG particles were removed and washed with PBS. Cells were stained with NucBlue live-ready probes (Hoechst 33342 dye) reagent (Invitrogen). The stain was removed after 10 min of incubation and washed with PBS. Live cell imaging solution was added to the cells and imaged using Evos FL Auto 2 (Invitrogen).