Anti cd47 percp cy5

Pancreatic tumour cell lines, MDDC and T-cells were assessed for expression of different markers using the following antibodies: Anti-PDL-1 APC, anti-Galectin 9 PE, anti-CD39 FITC, anti-CD47 Percp-Cy5.5, anti-HLA-class I Alexa Flour 700, anti-MIC A/B PE, anti-ULBP1 FITC, anti-ULBP 2,5 6 Percp, anti-ULBP 3 APC,anti-CD86 PE, anti-CCR7 PE-CY7, anti-CD40 APC, anti-HLA-class II PE-CY5 anti-IFN-γ PE-CY7, anti-CD3 Alexa Flour 488, anti-CD4 APC-CY7, anti-CD8-PE and anti-CD69 APC (Biolegend, CA).

Between days 13 and 17, cells were dissociated into single cells using 0.05% trypsin-EDTA (Gibco, catalog #25300062) at 37 °C for 10–12 min. Cells were then washed in an equal volume of “Stop” medium, which contained 10% fetal bovine serum (Gibco, catalog #16141079), and centrifuged at 300×g for 5 min. For cell lines containing NKX2-1:GFP reporter (“C17” and “BU3”), cells were directly resuspended in “Sort buffer,” composed of Hanks Balanced Salt Solution, HEPES, EDTA, FBS, and Y-27632, and passed through a 40-µm cell strainer (Fisher, catalog #087711). In all, 10 µM calcein blue AM (Life Technologies, catalog #1429) was added and the cells were sorted for NKX2-1^GFP using a high-speed cell sorter (MoFlo Astrios Cell Sorter). Cell lines without NKX2-1:GFP reporter (“BU1”, “RUES2”, “RC2 204”, “CFTR4-2”, “P20801”, “CFTR5-5,” “W1282X #2,” “G551D #3”) were resuspended in “Sort buffer” at a concentration of 1×10⁶ cells/100 µL and stained with anti-CD47-PerCP Cy5.5 (Biolegend, catalog #323110) and anti-CD26-PE antibodies (Biolegend, catalog #302706) (or anti-CPM antibody) at 4 °C for 30 min. The cells were then washed with 1% fetal bovine serum, centrifuged at 300×g for 5 min, and resuspended in “Sort buffer” containing 10 µM calcein blue AM, prior to cell sorting for CD47^hi/CD26^neg cells as previously described^{28 (link)}.