Apc anti mouse cd137

InVivo MAb anti-mouse 4-1BB (clone: 3H3) and InVivoPlus rat IgG2a isotype control (clone: 2A3) were supplied by BioXCell (New Hampshire, USA). IRDye® 680RD NHS Ester Infrared Dye was supplied by Li-cor (Nebraska, USA). Fixable Viability Stain 780 was purchased from BD Pharmingen (San Diego, USA). Dimethyl sulfoxide (DMSO) Hybri-Max (TM) sterile-filtered BioReagent was purchased from Sigma (St. Louis, MO, USA). The AbC™ total antibody compensation bead kit was acquired from Thermo Fisher (Waltham, MA, USA). PE anti-mouse CD4, FITC anti-mouse CD8a, APC anti-mouse CD137, and Alexa Fluor 594 goat anti-rat IgG (minimal x-reactivity) antibodies were supplied by BioLegend (San Diego, CA, USA). Anti-rabbit IgG (H + L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) was purchased from Cell Signaling Technology (Massachusetts, USA). A purified anti-mouse CD3 antibody was supplied by Thermo (Waltham, MA, USA). An anti-CD137 antibody was purchased from Abcam (Cambridgeshire, UK).

Cells were stained for flow cytometry using CD3, CD4, and CD8 antibodies as well as dead cell marker for T-cells, and CD86, CD64, and CD11c for dendritic cells and macrophages. T-regulatory cells were fixed and stained using the T reg detection kit Miltenyi Biotec) according to manufacturer’s specification.
Antibodies used for flow were as follows: α-CD4 PE (Miltenyi Biotec), α-CD8 Vivobright FITC (Miltenyi Biotec), α-CD3 APC (MIltenyi Biotec), α-CD11c FITC (MiItenyi Biotec), α-CD86-PE (Miltenyi Biotec), and α-CD64-APC (Miltenyi Biotec).
Danger-Associated Molecular Patterns were analyzed by staining targeted cells with anti-human Calreticulin (CRT)-A647 (Bioss), anti-human HSP70-A647 (Bioss), anti-human GRP94-A647 (Bioss), and anti-human HMGB1-A647 (Bioss). yH2AX was analyzed by staining the cells using anti-human yH2AX (Biolegend). All antibodies were cross-reactive with mice.
To measure immunomarkers, the cells were stained with APC anti-mouse IFNAR-1 (Biolegend), APC anti-mouse CD252 (Biolegend), APC anti-mouse CD54 (Biolegend), APC anti-mouse CD152 (Biolegend), APC anti-mouse CD274 (Biolegend), APC anti-mouse CD137 (Biolegend), APC anti-mouse CEACAM-1/CD66a (R&D), Alexa Fluor 647 anti-mouse B7-H2 (R&D) Alexa Fluor 647 anti-mouse TRAILR1/TNFRSF10A (Novus), Alexa Fluor 647 anti-mouse CD27 ligand/TNFSF7/CD70 (Novus).
All cells were analyzed on a Guava Easycyte 8HT flow cytometer.