Apc cy7 conjugated cd45

Blood, spleen and bone marrow cells were analysed before RBC lysis. Briefly, total blood (2.5 µl diluted in 500 µl RPMI) and spleen and bone marrow (100 µl from a stock solution of 10⁷ cells/ml) were incubated (20 min, 4°C) with Fc-gamma blocking antibody (2.4G2, BD Biosciences) and further stained with phycoerythrin (PE)-conjugated anti-Ter-119, fluorescent isothiocyanate (FITC)-conjugated anti-CD71, Allophycocyanin (APC)-conjugated CD44 (eBioscience, ImmunoSource), PE-Cy7 conjugated rat anti-CD11b (BD Pharmingen) and matching control antibodies. The cells were washed with PBS, measured on FACSCanto II (BD Biosciences) and the results analysed using FlowJo software by gating on Ter-119⁺ cells.
The bone marrow, spleen and liver cells after RBC lysis were analyzed using APC-Cy7 conjugated CD45 (BD Pharmingen), PE-Cy7 conjugated rat anti-CD11b (BD Pharmingen), Alexa647-conjugated rat anti-Ly6c (Serotec), PE-conjugated rat anti-Ly6G (BD Pharmingen) and matching control antibodies. 7AAD (BD Pharmingen) was used to exclude death cells.

After being blocked with CD16/CD32 (Cat. 553,141, BD Pharmingen™, USA), single-cell suspension (1 × 10⁶ cells) was incubated with a flow panel for 30 min avoiding light, the panel included APC-CY7 conjugated CD45 (Cat. 557,659, BD PharmingenTM, USA), PE-conjugated F4/80 (Cat. 565,410, BD PharmingenTM, USA), APC-R700 conjugated CD86 (Cat. 565,479, BD PharmingenTM, USA), and AF-647 conjugated CD206 (Cat. 565,250, BD PharmingenTM, USA). The ratio of Panc02 cells to M0 macrophages was then calculated using flow cytometry (CytoFLEXS, Beckman COULTER, USA) to identify the single-cell suspension.