Anti epcam percp cy5

Single cell suspensions were incubated with primary antibodies and isotypic controls, followed by Alexa Fluor dyes-conjugated secondary antibodies, or stained with fluorescence-conjugated primary antibodies: anti-EpCAM-PerCP-CY5.5 (BD Biosciences), anti-CD44-APC (BioLegend), anti-CD133/1-PE (Miltenyi Biotec), anti-CD166-PE (R&D Systems), anti-CD146 monoclonal antibody (AA1, made in our laboratory). Flow cytometry analyses were performed using BD FACS Calibur. Cell sorting was conducted using FACS Aria Cell Sorting System (BD).

Tumor samples were digested using half the amount of reagent required per sample of Miltenyi Biotech Liver Kit as per the manufacturer's instructions in C-tubes on a gentleMACS™ dissociator (Miltenyi Biotech) with heaters. After digestion, samples were filtered through Falcon filter cap tubes and washed with 2 ml Dulbecco's modified Eagle medium (Gibco). Samples were centrifuged at 500 g for 5 min, washed twice with AutoMACS Rinsing Solution (Miltenyi Biotech) with 1% bovine serum albumin and 0.5 mM EDTA, and resuspended in 1 ml of the aforementioned solution for counting and viability check on an Attune NxT cytometer. Cells (10⁶) were incubated with the following antibodies: anti-CD16/32 APC-R700, anti-CD11b APC, anti-F4/80 PE-Cy7, anti-EpCAM PerCP-Cy5.5, anti-CD45 BV605, anti-CD11c FITC, anti-NKp46 PE (BD Biosciences; diluted at 1:000 to 1:10.000). Data were acquired on a BD Biosciences FACSymphony A5 cytometer and analyzed with FlowJo Software (BD Biosciences).

Flow cytometry and CSC gating were performed as previously described [28 ]. Briefly, cells were stained with anti-CD44-APC (559942; BD Biosciences, Franklin Lakes, NJ), anti-CD24-PE (555428; BD Biosciences), anti-EpCAM-PerCP-Cy5.5 (347199; BD Biosciences) antibodies, Hoechst 33258 (BD Biosciences), propidium iodide (PI; 556463; BD Biosciences), and Annexin V (Biolegend, San Diego, California; 640918). Flow cytometric analysis was performed on single cell suspensions for the CD44^hi/CD24^lo/EpCAM⁺ CSC phenotype.