X 20 flow cytometer

In a separate Tu-2449SC subcutaneous tumor model experiment, mice from each assigned group that showed measurable tumor growth were sacrificed at day 39 post tumor implantation. Tumors were excised from the skin and minced into small pieces in HBSS (ThermoFisher Scientific, # 14175095) and single-cell suspensions were obtained using gentleMACS™ C Tubes with gentleMACS™ Dissociator (Miltenyi Biotec, # 130-096-334 and # 130-093-235), and enzymatic solution containing 50 µg/mL final Liberase™ TM (Sigma-Aldrich, # 5401119001) and 200 U/mL final Deoxyribonuclease I (Sigma Aldrich, # D5025). After filtration through a 70 µm cell strainer and washing, erythrocytes were removed following incubation with eBioscience 1X RBC Lysis Buffer (ThermoFisher Scientific, # 00-4300-54). Viable cell counts were determined using a Cellometer K2 (Nexcelom Bioscience). For subsequent flow cytometry analyses, cells were first incubated with TruStain FcX antibody (BioLegend, # 101320) and Zombie UV (BioLegend, # 423108) before being subjected to surface staining with fluorochrome-conjugated antibodies against mouse CD45 (BioLegend, # 103132), CD3ɛ (BD Biosciences, # 563565), CD4 (BD Biosciences, # 564922) and CD8α (BioLegend, # 100759). Acquisition was conducted on an LSRFortessa X-20 flow cytometer and analysis by using FlowJo software (version 10) with flowAI (1.7) plugin as described [52 (link)].