Portions of primary tumor and metastatic tissues were digested with collagenase IV/DNase (0.2 mg/mL and 10 μg/mL) at 37 °C for 30 min. Cells were put through 40-μm filters to obtain single-cell suspensions and stained as previously described [21 (link)]. Cell surfaces were stained with anti-mouse CD4, anti-mouse CD206, anti-mouse CD11b, anti-mouse F4/80, anti-mouse Gr1.1, anti-mouse CD3, anti-mouse CD45, anti-mouse CD8a, anti-mouse CD28, anti-mouse NKG2D, anti-human CD45, anti-human CD8, anti-human CD33, and anti-human HLA-DR antibodies (BioLegend, San Diego, CA) labeled with fluorescein isothiocyanate, PerCP/Cy5.5, phycoerythrin, or Brilliant Violet 421. Flow cytometry was performed using an Attune flow cytometer (Life Technologies), and the data were analyzed with FlowJo software.
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