Fitc conjugated anti mouse cd25

Monoclonal antibodies (mAbs) used included PE and/or FITC-conjugated anti-mouse CD4 (BD Pharmingen), FITC-conjugated anti-mouse CD8 (BD Pharmingen), Percp-conjugated anti-mouse CD3 (BD Pharmingen), and FITC-conjugated anti-mouse CD25. BALF cells (1 × 10⁵) were stained with an mAb for 30 min on ice, washed, and quantified using FACScan (Becton-Dickinson Immunocytometry System, San Jose, CA, USA).

For detection of myeloid derived suppressor cells (MDSCs), peripheral blood cells from DC vaccine-immunized and control mice were collected, and cells were stained for 30 min at 4°C with antibodies against specific cell markers, including FITC-conjugated anti-mouse CD11b (for cell surface), APC-Cy7 conjugated anti-mouse Ly-6C and PE conjugated anti-mouse Ly-6G, (both for intracellular staining). All three antibodies were obtained from Biolegend, (San Diego, CA). The percentages of monocytic and granulocytic MDSCs were gated on CD11b⁺Ly-6C⁺ cells and CD11b⁺ Ly-6G⁺ cells, respectively.[19 (link)] For Treg cell detection, cells were surface stained with FITC-conjugated anti-mouse CD25 and inter-cellularly stained with APC conjugated anti-mouse Foxp3, followed by cell permeabilization with the Cytofix/Cytoperm Plus kit according to the manufacturer’s protocol for all three tested antibodies, and the reagent kit from BD pharmingen (San Diego, CA). Percentage of Treg cells was gated on CD25⁺ Foxp3⁺ cells.[20 (link)] Fluorescence signals were detected by cytometry as described above.