Anti cd16 cd32 ab

For the assessment Treg cell populations by FACS, DLNs were harvested at 6 days following final viral treatment of 4T1 tumor-bearing mice. Cells were pretreated with saturating anti-CD16/CD32 Ab (Biolegend, San Diego, CA) in staining buffer (2% FBS, 0.02% sodium azide in PBS) to block cellular Fc receptors. Cells were stained extracellularly with peridinin chlorophyll protein-CY5.5-conjugated anti-CD4 Ab (BD Biosciences) and phycoerythrin-conjugated anti-CD25 Ab (eBioscience, San Diego, CA). Cells were then permeabilized with Foxp3 fixation/permeabilization buffer (eBioscience) according to the supplier's protocol and stained with allophycocyanin-conjugated anti-Foxp3 Ab (eBioscience). Samples were analyzed using a BD Biosciences BD FACScanto II flow cytometry analyzer and FACSDiva software (BD Biosciences).

For the determination of leukocyte subtypes and expression of surface markers, exudate cells were first blocked with anti-CD16 and anti-CD32 antibodies to prevent non-specific staining. The cells were stained with FITC-conjugated anti-mouse Gr-1 (0.5 µg/10⁶ cells), FITC-conjugated anti-mouse Ly6G (0.2 μg/10⁶ cells), PE-conjugated anti-mouse F4/80 (0.2 µg/10⁶ cells), PerCP-conjugated anti-mouse CD11b (0.2 µg/10⁶ cells), PB-conjugated anti-mouse Ly6C (0.2 μg/10⁶ cells), PE/Cy7-conjugated anti-mouse TLR-4 (0.5 μg/10⁶ cells), and APC-conjugated anti-mouse Tim4, CD206, or CD163 (0.3–0.5 μg/10⁶ cells). For intracellular staining of Gal-1, cells were blocked with anti-CD16/CD32 Ab (Biolegend) first, and then stained for surface markers and fixated with 4% PFA in 5% sucrose/PBS for 15 min at RT, followed by permeabilization with 0.01% tween-20 in 1% BSA in PBS for 20 min on ice. Then, the cells were incubated with mouse anti-Gal-1 (0.5 μg/10⁶ cells) or without primary antibody and then with Alx647 or Alx488-anti-mouse IgG (isotype control) for 20 min at RT, washed with 1% BSA in PBS. Stained cells were analyzed using FACSCalibur or FACSCantoII (BD Biosciences). Data analysis was performed using the FlowJo software.