Pacific blue antimouse cd45

CT26 tumors were harvested and single-cell suspensions were prepared using the Tumor Dissociation Kit (Miltenyi Biotec) with the gentleMACS Octo Dissociator (Miltenyi Biotec). 1 × 10⁶ cells were suspended in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin and the cells were transferred to six-well plates. Then 0.2 μg ODN1826-CY5 or 2 μg CCMV-ODN1826-CY5 were added and the cells were incubated at 37 °C in a 5% CO₂ atmosphere for 1 h. The cells were harvested and treated with Fc-block (BioLegend), then stained with Zombie Aqua (BioLegend). The cells were then washed in fluorescence-activated cell sorting (FACS) buffer (2% fetal bovine serum in PBS), and then stained with the following antibodies: Pacific Blue antimouse CD45, FITC antimouse CD11b, and PE antimouse F4/80 (all from BioLegend). After washing with FACS buffer, cells were acquired for immediate analysis using a BD LSR II flow cytometer (BD Biosciences). Data were analyzed using FlowJo v8.6.3 software. Tumor cells were gated as CD45⁻ cells, and TAMs were gated as CD45⁺ CD11b⁺ F4/80⁺ cells.