Diva software v 9

Macrophages were mechanically harvested after 7 to 12 days of differentiation/treatment using a cell scraper (Greiner Bio-one, Kremsmünster, AT), spun at 1000 rpm/10 min, and analyzed for cell integrity and viability by microscopy using Trypan blue exclusion or a Live/Dead cell viability dye (Ghost Dye^TM Violet 510, Tonbo Biosciences, San Diego, CA, USA). For immunophenotyping, viable cells were stained using specific fluorophore-conjugated anti-human monoclonal antibodies (Table 1) at concentrations recommended by the manufacturer. Cells were incubated with Abs for 30 min at +4 °C in the dark, washed using PBS/0.1% BSA (FACS Wash) buffer, and fixed in 4% Paraformaldehyde (Biolegend, San Diego, CA, USA) before analyzing at BD FACS Verse^TM flow-cytometer (BD Biosciences, San Diego, CA, USA). Live/dead dye was routinely applied to exclude dead cells before analyzing cells of interest. Data were analyzed using FACS Suite or Diva software v 9.0 (BD Biosciences, San Diego, CA, USA).

The membrane-associated markers CD133 and CD326/EpCAM were measured by flow cytometry with the following fluorochrome-conjugated antibodies purchased from Miltenyi Biotech (Bergisch-Gladbach, Germany): CD326/EpCAM-APC, human (clone: REA764, #130-098-118) and CD133/1 (AC-133)-PE (#130-098-826). Isotype antibodies were used as controls. Prior to antibody binding, cells were grown to confluency and maintained in medium with 0.5% FBS for at least one week to reduce the effect of serum. Following detachment with Biotase (Merck/Biochrom, Darmstadt, Germany and cell counting, 100,000 cells of PANC-1^RAC1-N17, PANC-1^RAC1b-KO, or the respective controls were incubated with antibodies diluted in phosphate-buffered saline (PBS)/3% human serum for 20 min. Analysis was performed on a FACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany) and Diva Software v9.0 (BD Biosciences).