Magnetic cd326 ep cam microbeads

After separation of blood using human peripheral blood lymphocyte separation liquid, mononuclear cells were washed twice with sterile Hank's balanced salt solution (Life Technologies, Shanghai, China). Isolated cells were enriched by binding to magnetic CD326 (Ep-CAM) MicroBeads (Miltenyi Biotech Ltd, Bergisch Gladbach, Germany) using magnetic activated cell sorting (MACS). Enriched isolated cells were then labeled with monoclonal antibodies targeting the epithelial cell antigens CD45, CD326 and cytokeratin 8, 18 and 19 (Miltenyi Biotech Ltd) and incubated in the dark at room temperature for 12 min. Antibodies specific for leukocytes (CD45) labeled with phycoerythrin (10 μL), specific for epithelial cells (cytokeratin 8, 18 and 19) labeled with fluorescein isothiocyanate (10 μL) and specific for epithelial cells (CD326/Ep-CAM) labeled with allophycocyan (10 μL) were added per 7.5 mL whole blood. Cell pellets were resuspended in 500 μL PBS and counted by flow cytometry using a BD FACSCanto™ II apparatus (Becton Dickinson, San Jose, CA, USA). Cells that were CD45 negative, CK positive and CD326 positive were defined as CTCs.

Peripheral blood sample (7 mL) was drawn 1 day before treatment and 1 month after the final transfusion for the detection of circulating tumor cell (CTC) levels. PBMC from the peripheral blood sample were separated using the human peripheral blood lymphocyte separation liquid (Haoyang Biological Manufacture Co., Ltd.) and were washed twice with sterile Hank’s balanced salt solution (Thermo Fisher Scientific, Waltham, MA, USA). Isolated cells were enriched via binding to magnetic CD326 (EpCAM) MicroBeads (Miltenyi Biotech Ltd., Bergisch Gladbach, Germany) using magnetic activated cell sorting. Enriched isolated cells were then labeled with monoclonal antibodies targeting epithelial cell antigens CD45, CD326, and cytokeratins 8, 18, and 19 (Miltenyi Biotech Ltd.) and incubated in the dark at room temperature for 12 min. Antibodies specific for leukocytes (CD45) labeled with phycoerythrin (PE) (10 μL), specific for epithelial cells (cytokeratins 8, 18, and 19) labeled with fluorescein isothiocyanate (FITC) (10 μL), and specific for epithelial cells (CD326/Ep-CAM) labeled with allophycocyan (10 μL) were added to 7.5 mL of blood. Cell pellets were resuspended in 500 μL PBS and counted by flow cytometry using a BD FACSCanto™ II apparatus (BD Biosciences). Cells that were CD45-negative, and CK- and CD326-positive were defined as CTCs.