Cd25 apc 3c7

Total blood cells or leukocytes from the liver or spleen were incubated with Fc block (eBiociences, Thermo Fisher Scientific) for 10 minutes at 4-8°C. Surface staining was performed for 20 minutes at 4-8°C and always included Fixable Viability Dye (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) for exclusion of dead cells. The following conjugated anti-mouse mAbs (and respective clones) were used: CD3 PerCP-Cy5.5 (145-2C11), CD4 APC or Brilliant Violet (BV) 510 (GK1.5), CD8 BV711 (53-6.7), CD62L FITC (MEL-14), CD19 PE (1D3), CD25 APC (3C7), CD44 PE-Cy7 (IM7), CD45 Alexa Fluor 700 (30-F11), CD69 BV650 (H1.2F3), CD127 PE-Dazzle 594 (A7R34), NK1.1 PE-Cy5 (PK136), KLRG1 PE (MAFA), CXCR3 APC (CXCR3-173), TCR γδ BV421 (GL3), all from either BioLegend (San Diego, CA, USA) or Sysmex (Kōbe, Hyōgo, Japan). Cells were acquired in a BD LSR Fortessa X-20 cytometer and analyses were performed within live, single (based on FSC-A vs. FSC-W parameters) CD45+ leukocytes using FlowJo v10 (FlowJo, BD). For the clustering and visualization of high-dimensional data, equivalent numbers of live, single CD45+ cells from each condition were concatenated. Clustering was performed using X-shift (number of clusters determined by the algorithm) and data are presented using the dimensionality reduction method TriMAP (large-scale dimensionality reduction using triplets).

Single-cell suspensions from mouse subcutaneous tumors were prepared as described above, counted, and resuspended in PBS at a concentration of 1×10⁷ live cells/mL. One hundred microliters of each sample was plated for cell staining. Surface staining was performed at room temperature for 30 min, and intracellular staining was performed using a Foxp3-transcription factor staining kit (eBioscience). Live/dead cell discrimination was performed using Fixable Viability Stain 520 (BD Biosciences). Anti-mouse antibodies against the following antigens were used for flow cytometry: CD3 PerCP-CY5.5 (17A2, BioLegend, 1:100), CD45 PerCP (30-F11, BioLegend, 1:200), NK1.1 APC (PK136, BD Biosciences, 1:50), Foxp3 PE (MF23, BD Biosciences, 1:100), Ly6G PE (1A8, BioLegend, 1:100), Ly6C APC (HK1.4, BioLegend, 1:100), CD11b BV421 (M1/70, BioLegend, 1:100), CD11b PerCP-CY5.5 (HL3, BioLegend, 1:100), F4/80 APC (T45–2342, BD Biosciences, 1:50), CD25APC (3C7, BioLegend, 1:100), CD4 APC (GK1.5, BioLegend, 1:100), and CD8a PE (53-6.7; BioLegend, 1:100). All flow cytometry analyses were performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).