Anti cd3 fitc 17a2

Groups of five C57BL/6 mice challenged with MOC7 cells under the immunotherapy protocol described above in the prophylactic bioassay were used to determine the presence of tumor-infiltrating lymphocytes and antigen-presenting cells (APCs), as described by Oyarce et al. [52 (link)] and Murgas et al. [53 (link)]. The mice were euthanized at day 30, and the tumors were removed. Single-cell suspensions of the tumors or organs were prepared using a solution containing collagenase IV (5 mg/ml, Gibco, Thermo Scientific) and DNase I (5 mg/ml, AppliChem, Maryland Heights, MO, USA) in RPMI supplemented with 0.5% FBS for 45 minutes at 37°C in a shaker bath. Then, the cells were stained with the following conjugated antibodies: anti-NK 1.1 Brilliant Violet 421 (PK136, BioLegend, San Diego, CA, USA), anti-CD-F4/80 PERCP (BM8, BioLegend), anti-CD11b APC (M1/70, BioLegend), anti-CD3 FITC (17A2, BioLegend), anti-CD4 PERCP (GK1.5, BioLegend), anti-CD8 APC/Cy7 (53-6.7, BioLegend), and Zombie Aqua (BioLegend) for 40 minutes at 4°C, then fixed with a 2% paraformaldehyde solution, and finally evaluated by flow cytometry (FACSCanto II, BD).

Single-cell suspensions were prepared by triturating the spleens between the ends of sterile frosted slides and filtration through nylon mesh. After erythrocyte lysis, splenocytes were suspended in RPMI 1640 tissue medium supplemented with 10% heat-inactivated foetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin, and cultured for 3 h at 37 °C with Cell Stimulation Cocktail (eBioscience, CA, USA). After FcγR blockade with anti-CD16/32 antibodies (BioLegend, CA, USA), extracellular antigens were stained for 20 min at 4 °C in RPMI 1640 medium. Cells were fixed and permeabilised using Foxp3/Transcription Factor Buffer Set (eBioscience) and stained for intracellular cytokines. Samples were acquired using a BD FACSCalibur flow cytometer operated by CellQuest (BD Biosciences, CA, USA), and the data were analysed using FlowJo software (BD Biosciences). Cell populations were identified by staining with anti-CD3-FITC (17A2), anti-CD4-PerCp/Cy5.5 (GK1.5), anti-IL-17A-PE (TC11-18H10), and anti-IFN-γ–Alexa Flour 647 (XMG1.2) (BioLegend).