Apc conjugated anti epcam antibody

Hepa1-6, HepG2, and Hep3B cells were cultured to 80% confluency by employing ATCC-recommended cultural methods in T75 tissue culture treated flasks (TPP, Switzerland). On the day of cell sorting, cells were trypsinized (Corning) and viable cells were determined using trypan blue and haemocytometer. Cells were stained using APC conjugated Anti-EpCAM antibody (Miltenyi Biotech, Germany) per manufacturer instructions in the dark. Before cell sorting, each cell preparation was filtered through a 40-µm sieve (VWR) and kept on ice. Cell sorting was performed using BD MoFlo cell sorter. Sorted cells were collected in complete media with 10% FBS, washed, and cultured back in fresh complete media with 10% FBS at 37°C incubator with 5% CO₂. To remove bound anti-EpCAM antibodies and bring cells back in log phase, sorted cells were grown for 3–4 days after sorting, and used for orthotopic injection.

HCC cells were prepared as single-cell suspensions for staining. All antibodies used for staining were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany), and included a phycoerythrin (PE)-conjugated anti-CD133 antibody (Cat #130-110-962), a PE-Vio770-conjugated anti-CD13 antibody (Cat #130-120-727), an allophycocyanin (APC)-conjugated anti-EpCAM antibody (Cat #130-111-000), an APC-Vio770-conjugated anti-CD44 antibody (Cat #130-113-339) and anti-REA control antibody (Cat # 130-113-438, 130-113-440, 130-113-434, 130-113-445). The percentages of CD133⁺, CD13⁺, EpCAM⁺, and CD44⁺ within the HCC cell population were determined according to the manufacturer’s instructions. In brief, 1 × 10⁶ cells were centrifuged and resuspended in 98 µL of buffer, 2 µL antibody was added, and the cells were incubated for 10 min in the dark at 4 °C. The cells were washed with 1 mL of buffer, centrifuged at 300 g for 10 min, and resuspended in 400 µL of buffer for detection. Data were acquired with a BD LSRFortessa. All samples were analysed in triplicate.