Goat anti mouse igg texas red

Following treatments, cells were washed with PBS. BMDMs were removed from culture wells using cell dissociation media (Gibco, 13151-014) and washed with 2% BSA in PBS. Cells were then resuspended in Fc Block (5 μg/mL) on ice for 5 minutes. BMDMs were stained with fluorescently conjugated antibodies against macrophage-specific surface proteins including SIRPα and Jurkat cells were stained with primary anti-CD47 monoclonal antibody (ThermoFisher, MA5-11895, clone B6H12.2) or isotype control mouse IgG1,κ (ThermoFisher, 14-4714-82) on ice for 20 minutes in the dark. Jurkats were then stained with goat anti-mouse IgG-Texas Red (JacksonImmuno, 115-076-071) for 15 minutes on ice. All cells were washed with PBS and resuspended in a Ghost viability dye (Tonbo Biosciences) for 20 minutes in the dark. To determine the extent of apoptosis induced in Jurkats, cells were stained with Annexin V (ThermoFisher, A13201) and propidium iodide following manufacturer’s instructions. Briefly, cells were resuspended in annexin-binding buffer and AF488-conjugated Annexin V was added to each sample and incubated for 15 min at room temperature. Stain was then diluted in more annexin-binding buffer and propidium iodide was added to samples. Cells were run on a cytometer soon after staining.

CD61⁺ cells in IMDM+20% BIT medium with 100 ng/ml Tpo and 100 ng/ml SCF were incubated with 90.9 μg/mL DQ Ova at 37°C or 4°C for 2 hours. To stop the reaction and remove excess DQ Ova, cells were washed three times with cold PBS + 0.5% FBS + 0.1% sodium azide. Cells were incubated with mouse anti-CD41a for 30 minutes, then incubated with goat anti-mouse-IgG-Texas Red (Jackson ImmunoResearch, West Grove, PA) for 30 minutes. Stained cells were fixed with 3.7% paraformaldehyde (Polysciences, Warrington, PA) in PBS for 10 minutes, then stained with DAPI for 10 minutes. Cells were allowed to settle on poly-D-lysine-coated glass slides for 1 hour, then a glass coverslip was mounted using Flouromount G (SouthernBiotech, Birmingham, AL). Slides were imaged using a 63X oil objective on an SP5 II laser scanning confocal microscope (Leica, Wetzlar, Germany).