Alexa fluor 488 conjugated hepcam antibody clone 9c4

BMC from tumor-bearing mice were obtained by density gradient centrifugation (see above), resuspended in DMEM + 10% FBS + 1% penicillin/streptomycin and transferred into cell culture dishes. 12 hours later, non-adherent cells were washed away with PBS and the adherent cells were kept in culture. Alexa Fluor 488-conjugated hEpCAM antibody (clone 9C4, Biolegend) was added and the colonies were checked for EpCAM expression, which was present in all colonies. The resulting CTC cell culture was expanded for 2 passages (1–2 weeks), after which 1 × 10⁶ cells were reinjected subcutaneously into both flanks of NSG mice. The mice were daily monitored for tumor development.

CTC and HCT116 cell lines were harvested, stained in FACS buffer (PBS, 1% fetal bovine serum) with Alexa Fluor 488-conjugated hEpCAM antibody (clone 9C4, Biolegend) according to the manufacturer's instructions and analysed on a standard BD FACSCalibur flow cytometer (BD Biosciences). An unstained sample of each cell line was used to determine the background fluorescence and ensure proper gating, the resulting data was analyzed with WinMDI Software v2.9.