Anti cd64 pe

Flow cytometry was performed on leukocytes isolated from organs as described above. Cells were washed in FACS buffer containing HBSS, 1% BSA, 4.17mM sodium bicarbonate, and 3.08 mM sodium azide and incubated for 15 minutes at room temperature with Fc Block (anti-CD16/32; Tonbo, San Diego, CA) except for stains for FcγRIIb. Cells were then incubated for 30 minutes at 4°C with the following antibodies: anti-CD11c-FITC (Tonbo), anti-CD11b-V450 (Tonbo), anti-CD45.2-APCCy7 (Tonbo), anti-CD64-PE (BD, San Jose, CA), anti-CD36-APC (BD), anti-MHC Class II-PerCP-Cyanine5.5 (Tonbo), anti-CD32b-PE (Thermofisher, Waltham, MA), anti-TCRβ-V450 (Tonbo), and anti-CD4-PeCy7 (Tonbo). All samples were washed, and either fixed in 2% paraformaldehyde (PFA) or permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA) for intracellular staining overnight at 4°C. Cells were then stained an additional 30 minutes with anti-FoxP3-FITC (eBioscience) followed by washing and fixation in 2% PFA. Samples were run using a MACSQuant Analyzer (Miltenyi, Auburn, CA) and analyzed using FlowJo software.

Carotid arteries and aortic arch were dissected from mice and single
cell suspensions were prepared by digestion in 37°C for 1hr in
collagenase buffer. Single cells were first incubated with LIVE/DEAD Fixable
Aqua Dead Cell Stain Kit (Molecular Probes, Cat# L34957) for 30 minutes at room
temperature to stain for dead cells. After washing, the cell suspensions were
labelled with the following fluorescently conjugated antibodies for 1 hour at
4°C: anti-CD11c-APC-Cy7 (BD Pharmingen, Cat# 561241),
Anti-MHCII-eFluor450 (Invitrogen, Cat# 48-5321-82), anti-CD45- Alexa Fluor 700
(Invitrogen, Cat# 56-0451-82), anti-CD3- PE/Dazzle 594 (BioLegend, Cat# 100245),
anti-CD64-PE (BD Pharmingen, Cat# 558455), Anti-CD11b-PerCP/Cy5.5 (BioLegend,
Cat# 101227). Flow cytometry was performed with Gallios Flow Cytometer at the CU
Cancer Center Flow Cytometry Shared Resource core facility. Kaluza flow
cytometry analysis software was used for data analysis.

Monitoring of clone 4 was done by staining with anti-CD64-PE, CD137L-PE and CD86-APC (BD Bioscience, San Jose, CA) and OKT3 loading was evaluated by staining with a PE-conjugated AffiniPure F(ab′)₂ fragment goat anti-mouse IgG (Jackson Immunology Research Laboratories, West Grove, PA). T cells were stained with anti-CD3-FITC (or PerCP-Cy5.5), CD8-PB (or APC-H7), CD16-PE, CD56-PE-Cy7 (or PE), CD27-APC, CD28-PE-Cy7, CD127-PE, BTLA-PE (clone J168), CD45RA-FITC (or V450), CD57-FITC (BD Bioscience), CD4-PerCP-Cy5.5, TIM3-APC (clone F38-2E2) (eBioscience), PD1-PerCP-Cy5.5 (clone EH12.2H7) and CCR7-PerCP-Cy5.5 (BioLegend, San Diego, CA). Cells were stained in 100 μL FACS buffer containing AQUA live/dead dye (Invitrogen) on ice for 30 minutes. For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences) and subsequently stained for granzyme B-PE, perforin-FITC (eBioscience), and in some cases also with cleaved caspase-3-PE (BD Biosciences). Data acquired using a FACScanto II cytometer (BD Biosciences) and analyzed using FlowJo v 7.6.5 (Treestar) with different subsets defined using size, viability, and “fluorescence minus one” (FMO) controls.