Alexa fluor 647 labeled igg2b

We determined the cell surface expression of ABCG2 in the stable HeLa cell lines and in transiently transfected HEK293 cells 48 h after transfection. Antibody labeling was performed in trypsinized cells with the ABCG2-specific 5D3 mouse monoclonal antibody (gift of Bryan Sorrentino, Division of Experimental Hematology, Department of Hematology/Oncology, St. Jude Children’s Research Hospital). Ko143 is an ABCG2 inhibitor that has an impact on ABCG2 conformation, thus helping 5D3 antibody recognition. 1 µM Ko143 (Sigma-Aldrich, cat. K2144) was added to the samples before the measurements. Alexa Fluor 647-labeled IgG2b (Thermo Fisher, cat. A-21242) was used as a secondary antibody. Propidium iodide (final concentration: 1.6 µg/ml, Sigma Aldrich, cat. 4170) was used for dead cell separation. Measurements were carried out in FACS Canto II after gating for live, EGFP positive cells.

We determined the cell surface expression of ABCG2 in transiently transfected HEK293 and HeLa cells 48 h after transfection, and an additional 24 hours after drug treatments. Cells were trypsinized and then the membrane localized ABCG2 was labeled by the 5D3 monoclonal antibody (gift of Bryan Sorrentino, Division of Experimental Hematology, Department of Hematology/Oncology, St. Jude Children’s Research Hospital) by adding 1 µM Ko143 inhibitor (Sigma Aldrich, cat. K2144) to achieve optimal antibody binding. Alexa Fluor 647-labeled IgG2b (Thermo Fisher, cat. A-21242) was used as a secondary antibody. Propidium iodide (final concentration: 1.6 µg/ml, Sigma Aldrich, cat. 4170) was used for separation of the dead cells. Measurements were carried out in a FACS Canto II equipment after gating for live cells.