*4.7. Flow Cytometric Analyses*

Tumors and adjacent healthy renal tissues were dissociated using the human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany, 130-095-929) and GentleMACS System (Miltenyi Biotec). Samples were acquired with a LSRII/Fortessa flow cytometer (BD, Heidelberg, Germany) expressed as mean fluorescence intensity (MFI). CompBeads (BD) were used for single color compensation to create multi-color compensation matrices. For gating, fluorescence minus one (FMO) controls were used. Prior to experiments, all antibodies and secondary reagents were titrated to determine optimal concentrations.

For staining of FPN, extracellular staining of patient-derived single cell suspensions was performed, containing CD33 BV510 (BD, 563257), MerTK BV421 (Biolegend, San Diego, CA, USA, 367603), CD45 AF700 (Biolegend, 368513), CD 64 BV605 (Biolegend, 305033), CD206 PE-Cy7 (Biolegend, 321124), CD326 PE-CF594 (BD, 565399), HLA-DR APC-Cy7 (Biolegend, 307658), and FPN PE (Novus, Wiesbaden, Germany, NBP1-21502).
