Anti cd33

The Vδ2 T cells and PMN-MDSC frequency and phenotype were evaluated utilizing the following monoclonal antibodies: anti-Vδ1 (Life technology), anti-NKG2A, anti-NKG2D (Beckman Coulter), anti-NKG2C (R&D system), anti-Vδ2, anti-CD3, anti-CD15, anti-CD33, anti-HLA-DR, cocktail of antibodies anti-CD3, -CD56, -CD19, anti-CD14, anti-CD11b (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 1% paraformaldehyde and analyzed using a FACS Canto II (Becton Dickinson). For intracellular staining, membrane staining was performed as above described. After fixation cells were incubated with anti-IFNγ (BD Biosciences) for 30 min at room temperature. CD107a detection was accomplished by antibody staining during cell stimulation. After washing cells were analyzed using a FACS Canto II (Becton Dickinson). Apoptosis induction of Daudi cells were accomplished by evaluating Annexin V ligation to Daudi (Annexin V-FITC Apoptosis Detection Kit, eBiosciences) following the manufacturer’s instruction. Then cells were stained with anti-CD19, anti-Vδ2, anti-CD3, anti-CD15.

Plasma and ex vivo PortalBMC culture conditioned media were added at 25% media volume to counted cell number cultures of the myeloid precursor cell line U937 (ATCC) to examine the potential for PDAC blood soluble growth factors to affect myeloid differentiation. After 7 days at 37°C/5%CO₂ in culture, the U937 cells were harvested with cell dissociation media (Sigma-Aldrich) at 4°C, 15-30min, and analyzed by flow cytometry for biomarkers of development of myeloid differentiation/ antigen presenting cell (APC) markers relative to those found on untreated U937 cells grown without conditioned media: CD14+ (BD Biosciences 340585)/MHCII DR+ (BD Biosciences 560743) for monocyte/macrophage APC, CD11c (BD Biosciences (561355)/MHCII DR+ for dendritic cell APC, and CD14+CD33+CD11c-DR- for myeloid-derived suppressor cells (MDSC, anti-CD33, BD BioSciences (551378), and CD14+CD105+ for myeloid-derived fibroblasts (M-FB, anti-CD105 BD Biosciences (562408, 561443)).

Cell lines were cultured (5 × 10⁵ cells/well) at the indicated concentrations of WIN-55 or DMSO in triplicate wells for 18, 48 and 72 h. Isolated primary cells were cultured (5 × 10⁵ cells/well) for 18 h with DMSO (control) or WIN-55 with or without CB antagonists. WIN-55 was added at different concentrations and time-points. Cell viability was determined by Cell Counting Kit (CCK-8) assay as per manufacturer’s instructions (Dojindo Molecular Technologies). Optical densities were measured at 450 nm using a plate reader MultiskanTM Go Microplate (Thermo Fisher Scientific, Waltham, MA, USA).
AML cells from patients’ BM were identified at diagnosis with a combination of monoclonal antibodies against AML cells-associated antigens at diagnosis (anti-CD33, anti-CD34, anti-CD117, and anti-CD45 [BD Biosciences]). Apoptosis was assessed by Annexin V/7AAD staining assay kit, as per manufacturer’s instructions (BD Biosciences) and analysed on a FACS Canto II Flow Cytometer (BD Biosciences) and analysed with Infinicyt^TM Software (Cytognos, Spain).
Other studies, such as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), western blot, mitochondrial damage or enzyme activity assays are detailed in the supplementary files.