Apc conjugated anti human cd326 epcam antibody

To detect CAR expression on cell surface, recombinant biotinylated protein L (Thermo Fisher, United States ) was used to bind to scFv for 30 min at 4°C, followed by phycoerythrin (PE) -conjugated streptavidin (eBioscience, Lot4306318, United States). To detect EpCAM expression on target cells, APC- conjugated anti-human CD326 (EpCAM) antibody (Biolegend, Cat369809, United States) was used. To detect phenotype of T cells, APC-conjugated anti-human CD3 antibody (Invitrogen, Lot1987999, United States), FITC-conjugated anti-human CD4 antibody (eBioscience, Lot 4309494, United States), PE-Cy7-conjugated anti-human CD8 antibody (invitrogen, Lot2071279, United States) were used. To analyse memory phenotype of the CAR-T cells, PerCP-conjugated anti-CCR7 antibody (Biolegend, Cat353241, United States) and PE-conjugated anti-CD45RA antibody (Biolegend, Cat304108, United States) were used. Cells were stained with antibodies in dark for 30 min at 4°C and washed with fluorescence activated cell sorting (FACS) buffer (PBS, 2% BSA) before and after staining. Flow cytometry data were acquired with a CytoFlex S (Beckman Coulter, United States) and analysed by Flowjo V10.

The cell aggregates were incubated in 0.25% Trypsin-EDTA (15400-054, GIBCO) at 37°C for 15 min. The dissociates were quenched by FBS, followed by pipetting to generate a single-cell suspension. To analyze or sort hPGCLCs with cell surface markers, samples were stained with APC-conjugated anti-human CD326 (EpCAM) antibody (324208, Biolegend) and BV421-conjugated anti-human/mouse CD49f (INTEGRINa6) antibody (313624, Biolegend) at 4°C for 15 min. The samples were loaded on a MoFlo XDP (Beckman Coulter).