Ad no apparent disease conditions [39,40]. In this report we show that

Ad no apparent disease conditions [39,40]. In this report we show that individuals heterozygous for the potentially miss-processed ABCG2 variant (Q141K) have significantly lower ABCG2 protein expression in their red cells than individuals carrying the wild-type ABCG2 gene. Moreover, heterozygous individuals with an ABCG2 nonsense mutation on one allele, have about 50 reduction in their red cell ABCG2 protein expression. These data suggest that determination of the ABCG2 protein expression in the erythrocyte membrane may provide clinically valuable information for assessing the role of this protein in relevant diseases, metabolic conditions, or the efficiency and/or toxicity of drug treatments.MethodsAnticoagulated blood samples of healthy volunteers (47 unrelated individuals and 14 family members of two probands selected from the donor cohort) were fixed in paraformaldehyde (PFA), stained with monoclonal antibodies specifically recognizing membrane proteins, and subjected to flow cytometry (FACS). In parallel, genomic DNA was isolated from the blood samples; common SNPs of the ABCG2 gene were screened by LightCycler allelic discrimination system, while mutations were determined by direct sequencing. All subjects gave their written informed consent to participate in the study. This study was approved by the regional ethical committees, and all procedures were performed in accordance with the Declaration of Helsinki.For staining the red cell membrane plasma membrane calcium pump (PMCA) protein we used the 5F10 monoclonal Lixisenatide antibody (final concentration 4 mg/ml, ABCAM, Ab2825), recognizing all 4 human PMCA isoforms having common peptide epitopes [43]. As shown in the Supplementary materials for the 5F10 monoclonal antibody, a calibration of a wide range of the applied antibody assured maximum labeling for the red cell protein. After washing out the primary antibodies, secondary antibodies corresponding to the IgG type and labeled with phycoerythrin (PE) were added to the cells (goat anti-mouse (GAM) IgG1-PE: Invitrogen P21129, GAM IgG2a-PE: Invitrogen P21139, GAM IgG2b-PE: Invitrogen, P21149, final concentrations 5 mg/ml), incubated for 30 min at 37uC, washed and resuspended in PBS. In parallel tubes cells were directly stained with FITC-conjugated anti-Glycophorin A (GlyA) monoclonal antibody (final concentration 5 mg/ml, Gly-A-FITC: Beckman Coulter, 2212). We have used several batches of the respective antibodies that all showed similar results at the described concentrations. Intact red cells and erythrocyte ghost were gated based on the forward 223488-57-1 web scatter (FSC) and side scatter (SSC) parameters. Both fractions were analyzed for antibody staining by a FACSCalibur flow cytometer (excitation wavelength: 23977191 488 nm (Argon ion laser) emission filters: 585/42 nm for PE, 530/30 for FITC ?for details see Fig. 1). A careful optimization for maximum antibody labeling was carried out for all monoclonal antibodies (see Results). For ABCG2 expression controls, we used K562 cells retrovirally transduced to express ABCG2, as described previously [44]. As described earlier [44], PFA fixation of the membrane ABCG2 protein eliminates the conformation-sensitivity of its interaction with the 5D3 antibody, and provides maximum labeling.Determination of Expression LevelsIn order to use a combination of all relevant anti-ABCG2 antibodies for quantifying ABCG2 expression in the red cell membranes, we calculated an average binding factor, based on the relative stainin.Ad no apparent disease conditions [39,40]. In this report we show that individuals heterozygous for the potentially miss-processed ABCG2 variant (Q141K) have significantly lower ABCG2 protein expression in their red cells than individuals carrying the wild-type ABCG2 gene. Moreover, heterozygous individuals with an ABCG2 nonsense mutation on one allele, have about 50 reduction in their red cell ABCG2 protein expression. These data suggest that determination of the ABCG2 protein expression in the erythrocyte membrane may provide clinically valuable information for assessing the role of this protein in relevant diseases, metabolic conditions, or the efficiency and/or toxicity of drug treatments.MethodsAnticoagulated blood samples of healthy volunteers (47 unrelated individuals and 14 family members of two probands selected from the donor cohort) were fixed in paraformaldehyde (PFA), stained with monoclonal antibodies specifically recognizing membrane proteins, and subjected to flow cytometry (FACS). In parallel, genomic DNA was isolated from the blood samples; common SNPs of the ABCG2 gene were screened by LightCycler allelic discrimination system, while mutations were determined by direct sequencing. All subjects gave their written informed consent to participate in the study. This study was approved by the regional ethical committees, and all procedures were performed in accordance with the Declaration of Helsinki.For staining the red cell membrane plasma membrane calcium pump (PMCA) protein we used the 5F10 monoclonal antibody (final concentration 4 mg/ml, ABCAM, Ab2825), recognizing all 4 human PMCA isoforms having common peptide epitopes [43]. As shown in the Supplementary materials for the 5F10 monoclonal antibody, a calibration of a wide range of the applied antibody assured maximum labeling for the red cell protein. After washing out the primary antibodies, secondary antibodies corresponding to the IgG type and labeled with phycoerythrin (PE) were added to the cells (goat anti-mouse (GAM) IgG1-PE: Invitrogen P21129, GAM IgG2a-PE: Invitrogen P21139, GAM IgG2b-PE: Invitrogen, P21149, final concentrations 5 mg/ml), incubated for 30 min at 37uC, washed and resuspended in PBS. In parallel tubes cells were directly stained with FITC-conjugated anti-Glycophorin A (GlyA) monoclonal antibody (final concentration 5 mg/ml, Gly-A-FITC: Beckman Coulter, 2212). We have used several batches of the respective antibodies that all showed similar results at the described concentrations. Intact red cells and erythrocyte ghost were gated based on the forward scatter (FSC) and side scatter (SSC) parameters. Both fractions were analyzed for antibody staining by a FACSCalibur flow cytometer (excitation wavelength: 23977191 488 nm (Argon ion laser) emission filters: 585/42 nm for PE, 530/30 for FITC ?for details see Fig. 1). A careful optimization for maximum antibody labeling was carried out for all monoclonal antibodies (see Results). For ABCG2 expression controls, we used K562 cells retrovirally transduced to express ABCG2, as described previously [44]. As described earlier [44], PFA fixation of the membrane ABCG2 protein eliminates the conformation-sensitivity of its interaction with the 5D3 antibody, and provides maximum labeling.Determination of Expression LevelsIn order to use a combination of all relevant anti-ABCG2 antibodies for quantifying ABCG2 expression in the red cell membranes, we calculated an average binding factor, based on the relative stainin.