Pe anti mouse cd3 clone 17a2

Single-cell suspensions of tumor-burdened lungs were re-suspended in FACS buffer (PBS, 1%BSA), blocked by non-specific staining with Fc block (anti-mouse CD16/32 mAb; BD Biosciences), The following antibodies were acquired from Biolegend and used in the flow cytometry analysis: BV605 anti-mouse CD45 (clone 30-F11), PE anti-mouse CD3 (clone 17A2), PE/Cy7 anti-mouse CD4 (clone RM4-5), BV711 anti-mouse CD8α (clone 53 − 6.7), BV421 anti-mouse Ly-6 C (clone HK1.4), PE anti-mouse Gr-1 (clone RB6-8C5), PE/Cy5 anti-mouse CD11b (clone M1/70), PE/Cy7 anti-mouse Ly-6G (clone 1A8), BV711 anti-mouse IFN-γ (clone XMG1.2), BV421 anti-mouse CD8α (clone 53 − 6.7), PE/Cy5 anti-mouse CD3 (clone 17A2). Flow cytometry data were acquired on a FACS Celesta flow cytometer and data was analysed with FlowJo V10.

The analysis was carried out as described previously [11 (link)]. In brief, the enzymatically digested (collagenase, hyaluronidase type IV-S and DNase I) pancreatic tissue samples (n = 3) were mashed through nylon mesh cell strainers (70 µm). Single-cell sample suspensions devoid of red blood cells were treated for 20 min/RT with antibodies, fluorescently labeled and analyzed (no less than 4 × 10⁴ cells) using flow cytometry (BD FACS Canto II) to quantify the percentage of CD4+ and CD8+ populations in pancreas and blood specimens. Antibodies (BioLegend, San Diego, CA, USA) were used according to the manufacturer’s instructions: PerCP/Cyanine5.5 anti-mouse CD45 (clone 30-F11), phycoerythrin (PE) anti-mouse CD3 (clone 17A2), fluorescein isothiocyanate (FITC) anti-mouse CD4 (clone GK1.5) and APC/Cyanine7 anti-mouse CD8a (clone 53-6.7).