Flow Cytometry-based ABCG2 Quantification in RBCs

The measuring of ABCG2 levels in RBCs were carried out according to a recently developed flow cytometry-based method [25 (link), 29 (link), 30 ]. Blood samples were freshly collected (2–6 h before the flow cytometry analysis). We fixed and permeabilized the RBC membranes by using 1% formaldehyde solution, resulting in RBC “ghosts”. WGA-Alexa Fluor 647 (Thermo Fisher, cat. W32466, final cc. 1 µg/ml) were added to the fixed and washed RBC ghosts to separate them from debris during flow cytometry. Ghosts were incubated with the ABCG2-specific primary antibody (Bxp-34, mouse monoclonal antibody, Abcam, cat. ab3379) followed by a secondary, Alexa Fluor 488-labeled goat anti-mouse (H + L) antibody (Thermo Fisher, A-11001), in 96 well plates. Cellular fluorescence was measured twice each case by a FACSCanto II flow cytometer equipped with a plate loader. The agreement between the two measurements were tested by intraclass correlation coefficient (ICC). ICC estimates and their 95% confident intervals were calculated based on an absolute-agreement, 2-way model (package irr in R). Intraclass correlation coefficient (ICC) showed strong significant agreement between the two measurements of Bxp34 (ICC = 0.988, 95% confident intervals were: 0.982–0.991, p < 0.001). Therefore, we used the average of the two results for further analysis.