Allophycocyanin conjugated anti foxp3 ab

To assess the Treg population by flow cytometry, the B16-BL6 tumors were harvested at 5 days following the final Tregs injection. Dissociated tumors were prepared as previously described [30 (link)]. Cells were stained as previously described [34 (link)]. Briefly, the cells were stained with peridinin chlorophyll protein-CY5.5-conjugated anti-CD4 Ab (cat. no. 550954, clone RM4-5, BD Biosciences), phycoerythrin-conjugated anti-CD25 Ab (cat. no. 12-0251-81, clone PC61.5, eBioscience, San Diego, CA, USA), and allophycocyanin-conjugated anti-Foxp3 Ab (cat. no. 17-5773-82, clone FJK-16s, eBioscience). Samples were analysed using a BD Biosciences BD FACScanto II flow cytometry analyser and FACSDiva software (BD Biosciences).

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).