Anti cd3 alexa fluor 700

Weighed tumors were minced and allowed to digest in a 2 ml mixture of collagenase (400 U type II collagenase, Worthington) and 0.2 mg/ml DNase I in RPMI media at 37°C for 1 hr. The mixture was gently vortexed every 10-20 minutes. The tissue lysate was filtered through a 40 μm mesh prior to immunostaining. The resulting single cell suspension was stained with fixable viability dye eFluor 780, anti-CD45.2 Pacific Blue, anti-CD3 PE-Cy7, anti-CD3 Alexa Fluor 700, anti-Foxp3 Alexa Fluor 700, anti-CD11c eFluor 615, anti-NK1.1 PE (all from eBioscience), anti-Granzyme B APC and anti-CD4 Qdot 605 (from Life Technologies), anti-CD8 Brilliant Violet 650, anti-CD11b Brilliant Violet 570, anti-CD19 Brilliant Violet 650, anti-F4/80 FITC (all from BioLegend), anti-Ly6C APC, anti-Ly6G PE-Cy7, and anti-Ki67 PE (BD Biosciences). The percent positive cells were analyzed by FlowJo and gated on CD45 positivity. To analyze the number of CD133⁺CD44⁺ cells, the single cell suspension was incubated in the dark, on ice with Aqua LIVE/DEAD Fixable Dead Cell Stain (Molecular Probes) 1:1000 for 30 min, followed by staining with anti-CD44 APC (eBioscience, 1: 400) and anti-CD133 PE (eBioscience, 1: 200). Unstained, LIVE/DEAD only, and single stain served as control. Doublets were gated out using forward scatter width/height and sideward scatter width/height event characteristics.

The following monoclonal antibodies were used for human FACS stainings: from BD Biosciences (San Jose, CA): anti-CD25 APC (2A3, 1:20), anti-CD45RO APC-H7 (UCHL1, 1:20), anti-CD4 V500 (RPA-T4, 1:20); from Biolegend (San Diego, CA): anti-CD45RA FITC (HI100, 1:20), anti-CD3 PerCP-Cy5.5 (HIT3a, 1:20), anti-CD127 PE-Cy7 (A019D5, 1:20), anti-CD8a Pacific Blue (RPA-T8, 1:50), anti-CD11b Pacific Blue (ICRF44, 1:50), anti-CD14 Pacific Blue (HCD14, 1:50), anti-CD19 Pacific Blue, anti-CD3 Alexa Fluor 700 (HIT3a, 1:20), anti-CD45 Alexa Fluor 700 (HI30, 1:20), anti-Ki67 APC (16A8, 1:200) or anti-Ki67 Brilliant Violet 605 (16A8, 1:400); from eBioscience (San Diego, CA): anti-Foxp3 Alexa Fluor 700 (PCH101, 1:100), anti-Foxp3 PE (236A/E7, 1:100); Unspecific binding of antibodies was prevented by incubation of cell suspensions with Fc-Block (Human TruStain FcX, BioLegend, 1:20) for 5 min at RT, followed by FACS staining for 20 min at RT in the dark. Cells were passed through a 40 μm cell strainer (NeoLab) to remove large debris.