Cd3 fitc clone 145 2c11

Cell suspensions from iliac lymph nodes, blood and tumors were first incubated with 1µg/10⁶ cells of Fc block treatment (BD Pharmigen) and then stained for surface markers in FACS buffer (PBS + 1% FBS). For Treg staining on cells isolated from iliac lymph nodes: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD3 FITC, clone 145-2C11 (Biolegend); CD4 Pacific Blue, clone RM4-5 (Biolegend); Foxp3 APC, clone FJK-16S (eBioscience). For Treg staining on cells isolated from tumors: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD45 PerCP-Cyanine 5.5 (eBioscience); CD4 Pacific Blue, (Biolegend); CD8 Brilliant Violet 510, clone 53-6.7 (Biolegend); Foxp3 APC, (eBioscience). For effector memory staining on cells isolated from blood: Live/Dead (Fixable Near-IR); CD8 PerCP, (Biolegend); CD44 APC (Biolegend); CD62L FITC, clone MEL-14 (BD Pharmigen).

Draining lymph nodes of mice were washed with 1% of penicillin/streptomycin
and 1% fungizone in PBS and mechanically dissociated on sterile Falcon
70 μm cell strainer. Lymph node cells were centrifuged 1300
rpm, 10 min, and counted. Cells of lymph nodes were analyzed by CyFlow
Space flow cytometer (Sysmex Partec, Gemany) for viability using propidium
iodide (PI; 2.5 μg/mL; Molecular Probes, USA) to detect necrotic
cells, and for phenotype by labeling cells for 20 min, at RT, with
the following antimouse antibodies: CD3 FITC (clone 145–2C11,
BioLegend, USA), CD19 PE (Miltenyi Biotec, Germany), CD11c PE (clone
N418, eBioscience, USA). Flow cytometry data were acquired following
standard guidelines^{82 (link)} and analyzed by the
FloMax software (Sysmex Partec, Germany). CD3+, CD19+, and CD11c+
were gated on a forward scatter (FSC) vs a side scatter (SSC) dot
plot. Gates were labeled according to FloMax software default (Figure S2B; gate “R1” includes
the whole reference cell population; gate “Q4” includes
CD3+, CD19+, or CD11c+ cells within the “R1” gate, whereas
“Q3” includes cells negative for all the above-mentioned
markers).