Anti rat igg alexa flour 488 conjugate

J774a.1 macrophages were seeded onto 18-mm glass coverslips #1.5 (Warner Instruments) at a density of 3 × 10⁵ cells per well in 12-well plates. Prior to the addition of bacteria, J774a.1 cells were incubated on ice for 30 min. Each well was then washed with cold DMEM. Approximately 350 μL of bacterial culture diluted into cold DMEM was placed onto the J774a.1 cells for a final MOI of 100. Cells were incubated on ice for 30 min while shaking horizontally followed by a 15-min static incubation. Each well was washed twice with cold HBSS to remove unbound bacteria and then fixed using 4% paraformaldehyde in phosphate-buffered saline. For detection by fluorescence microscopy the following steps were taken: block with 3% bovine serum albumin (BSA) in PBS for 1 h at RT, probe for GN050 with anti-GN050 OM polyclonal antibody at 1:150 in 3% BSA for 1 h at RT, wash, followed by anti-rat IgG Alexa Flour (AF)-488 conjugate (Thermo Scientific) at 1:1,500 in 3% BSA for 1 h at RT, probe for J774a.1 membrane (wheat germ agglutinin AF-594 [Thermo Scientific] at 5 μg/mL) and nucleus (Hoechst 33342 at 5 μg/mL) in HBSS for 10 min at 37°C, and mount the slides using ProLong Gold antifade with DAPI (Thermo Scientific) and allow to cure overnight, covered at RT. GN050 signal intensity was quantified using CellProfiler software over 40 to 60 fields of view.

J774a.1 macrophages were seeded onto 18-mm glass coverslips #1.5 (Warner Instruments) at a density of 3 × 10⁵ cells per well in 12-well plates. Prior to the addition of bacteria, J774a.1 cells were incubated on ice for 30 min. Each well was then washed with cold DMEM. Approximately 350 μL of bacterial culture diluted into cold DMEM was placed onto the J774a.1 cells for a final MOI of 100. Cells were incubated on ice for 30 min while shaking horizontally followed by a 15-min static incubation. Each well was washed twice with cold HBSS to remove unbound bacteria and then fixed using 4% paraformaldehyde in phosphate-buffered saline. For detection by fluorescence microscopy the following steps were taken: block with 3% bovine serum albumin (BSA) in PBS for 1 h at RT, probe for GN050 with anti-GN050 OM polyclonal antibody at 1:150 in 3% BSA for 1 h at RT, wash, followed by anti-rat IgG Alexa Flour (AF)-488 conjugate (Thermo Scientific) at 1:1,500 in 3% BSA for 1 h at RT, probe for J774a.1 membrane (wheat germ agglutinin AF-594 [Thermo Scientific] at 5 μg/mL) and nucleus (Hoechst 33342 at 5 μg/mL) in HBSS for 10 min at 37°C, and mount the slides using ProLong Gold antifade with DAPI (Thermo Scientific) and allow to cure overnight, covered at RT. GN050 signal intensity was quantified using CellProfiler software over 40 to 60 fields of view.