2.11. Formalin-Fixed and Paraffin-Embedded (FFPE) Dissociation and Flow Cytometry Analysis

To analyze tumor-associated macrophages (TAMs) and tumor-infiltrating immune lymphocytes from orthotopic PDAC arose in IL17A knock out mice or in wild-type mice treated or not with an anti-IL17A mAb, two 50 μm sections from a FFPE block were transferred into a gentle MACS C tube to be dissociated while preserving intact cells with the FFPE Tissue Dissociation Kit (130-118-052, Miltenyi Biotec, Bologna, Italy) following the manufacturer’s instructions. Five mice per group were analysed. Single cell suspensions were resuspended in PBS containing 0.5% BSA and 0.01% NaN₃, and unspecific binding sites were blocked by 10 min incubation with anti-CD16/CD32 mAb (BioLegend, clone 93). Staining was performed using the following antibodies: FITC anti-CD11b (Miltenyi Biotec, clone M1/70.15.11.5), PerCP anti-CD45 (Miltenyi Biotec, clone 30F11), FITC anti-CD4 (Miltenyi Biotec, clone REA604), Per-CP anti-CD8 (eBioscience, clone 53-6.7), AF647anti-Granzyme-B (51-8898-82 eBioscience, clone NGZB), APC anti-CD206 (141712 BioLegend, clone C068C2), APC anti-FoxP3 (17-5773-82 eBioscience, clone FJK-16s) and APC anti-IFNγ (17-7311-82 eBioscience, clone XMG1.2). Cells were washed again with 0.5% BSA plus 0.01% NaN₃ in PBS. For IFNγ, Granzyme-B and FoxP3 staining, cells were stained for surface markers, then fixed and permeabilized at 4 °C for 30 min with the Fixation Permeabilization kit (eBioscience) after several washes with 0.5% BSA plus 0.01% NaN₃ in PBS. After washing with permeabilization buffer, antibodies against IFNγ, Granzyme-B and FoxP3 were added for 20 min at 4 °C. All flow cytometry data were acquired using the Accuri™ C6 cytometer (BD Biosciences, Milan, Italy) and analyzed with FlowJo_vX.0.7 software (Tree Star from BD Biosciences).