Ghost viability dye

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.

Single-cell suspensions prepared above were pelleted and incubated with anti CD16/anti-CD32 (BD Biosciences; 2.4G2) in PBS. Cells were washed and stained with Ghost Viability dye (Tonbo Biosciences) in PBS. Following a wash in PBS, cells were stained for surface markers in PBS. For intracellular staining, cells were fixed and permeabilized with a FoxP3 buffer set (eBiosciences). Samples were run on a Fortessa analyzer (BD Biosciences) in the UCSF Flow Cytometry Core and collected using FACS Diva software (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (Treestar). Fluorophore-conjugated antibodies specific for mouse surface and intracellular antigens were purchased from eBiosciences, BD Biosciences and Biolegend. The following antibodies and clones were used: anti-Ly6G (1A8), anti-CD11b (M1/70), anti-CD11c (HL3), anti-MHC Class II, (M5/114.15.2), anti-Ly6C (HK1.4), anti-IL-17 (TCA11–18H10.1), anti-IL-4 (11B11), anti-IL-13 (eBio13a), anti-IFN-γ (XMG1.2), anti-CD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (SK1), anti-TCRγδ (GL3), anti-Gata3 (TWAJ), IL-10 (JES3), anti-CD45 (30-F11), and anti-FoxP3 (FJK-16s).