70 m pore cell strainer

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).