Anti mouse cd45 viogreen

Six to eight mm mean diameter tumors from neuT and neuT-pfpKO mice were minced with scalpels and then incubated with 1 mg/mL collagenase IV (Sigma-Aldrich) in RPMI-1640 (Life Technologies, Monza, Italy) at 37 °C for 1 h in an orbital shaker. The cell suspension was washed in PBS supplemented with 2% FBS (Invitrogen), incubated in a buffer for erythrocyte lysis (155 mM NH₄Cl, 15.8 mM Na₂CO₃, 1 mM EDTA, pH 7.3) for 10 min at RT, and then washed in RPMI-1640 supplemented with 10% FBS (Invitrogen). The cell suspension was passed through a 70-µm pore cell strainer (BD Biosciences, Milano, Italy), centrifuged (1400 rpm for 10 min), and re-suspended in a buffer for erythrocyte lysis. After 10 min of incubation at RT, tumor-infiltrating leukocytes were washed with RPMI-1640 supplemented with 10% FBS (Invitrogen), centrifuged (1400 rpm for 10 min), re-suspended in PBS, treated with Fc receptor blocker (anti CD16/CD32; BD Biosciences, Milano, Italy), and stained with the following antibodies: anti-mouse CD45 VioGreen, anti-mouse CD3 FITC, anti-mouse CD49b PE, anti-mouse CD4 APC Vio770, anti-mouse CD8 VioBlue, anti-mouse γδ TCR PE/Cy7, anti-mouse CD11b FITC, anti-mouse F4/80 APC, and anti-mouse GR-1 PE (Miltenyi Biotech, Milano, Italy) [27 (link)]. Samples were acquired and analyzed on a CyAn ADP (DakoCytomation, Milano, Italy) using Summit 4.3 software (DakoCytomation, Milano, Italy).

Tumorsphere-derived cells were stained with the Aldefluor kit (Stem Cell Technologies, Grenoble, France) as reported [51 (link)]. To measure intracellular ROS content, cells were stained with 2′, 7′-dihydrochlorofluorescein diacetate (DHCF-DA, Sigma-Aldrich) as described [9 (link)]. The results were expressed as percentages of positive cells and as mean fluorescence intensity (MFI). For the analysis of immune infiltrates, lungs and tumors from vaccinated mice were finely minced with scissors and then digested by incubation with 1 mg/mL of collagenase IV (Sigma Aldrich) in RPMI-1640 (Life Technologies) at 37 °C for 1 h in an orbital shaker. After washing in PBS, cell suspensions were incubated with a buffer for erythrocyte lysis (155 mM NH₄Cl, 15.8 mM Na₂CO₃, 1 mM EDTA, pH 7.3) for 10 min at room temperature, then passed through a 40 µm-pore cell strainer, centrifuged at 1800 rpm for 10 min and suspended in PBS. The cells were treated with an Fc receptor (FcR) blocker (CD16/CD32; Becton Dickinson) for 5 min at 4 °C and then stained for 30 min at 4 °C with the following Abs: anti-mouse CD45 VioGreen, anti-mouse CD3 FITC, anti-mouse CD4 APC-Vio770, anti-mouse CD8 VioBlue, and anti-mouse CD49b PE (all from Miltenyi Biotec, Bologna, Italy). All samples were acquired on a BD FACSVerse and analyzed with the BD FACSuite™ software (Becton Dickinson, Milan, Italy).