Efluor 506 conjugated anti mouse cd3 clone 17a2

Cell cycle analysis was performed on cells collected from single wells of a six-well tissue culture plate using 0.25% trypsin and washed in PBS prior to fixation with ice-cold 70% ethanol and stored at −20 °C for at least 8 hours. Following fixation, cells were again washed in PBS and then re-suspended in a solution of PBS containing 50 ug/mL propidium iodide (Life Technologies), and 100 ug/mL RNase A (Roche). For flow cytometry analysis of CD8+ T cells, tumors were minced in 2mg/ml collagenase IV followed by a 1-hour incubation at 37°C and then passed through a 70μm cell strainer, washed and pellet. Cells (1 × 10⁶) were then incubated with purified anti-mouse CD16/32 (Biolegend) and subsequently stained with the following monoclonal antibodies against mouse markers: eFluor 450-conjugated anti-mouse CD45 (clone 30-F11, eBioscience), eFluor 506-conjugated anti mouse CD3 (clone 17A2, eBioscience), FITC-conjugated anti-mouse CD8a (clone 53–6.7, Invitrogen) and PE-Cyanine 7-conjugated anti-mouse CD4 (clone GK1.5, eBioscience). Cells were analyzed on a FACSCanto flow cytometer using BD FACSDiva software (BD Biosciences) and data analyzed using FlowJo v10 (FlowJo, LLC).