Heterogeneity and kinetics of epidermal growth factor receptor (EGFR) expression in mammary epithelial cells (MECs). (A) Dual staining of aldehyde dehydrogenase 1 (ALDH1)-positive cells (green fluorescence) with anti-EGFR antibodies (top) or rhodamine (Rh)-labeled epidermal growth factor (EGF) (bottom) in human MEC-human telomerase reverse transcriptase (hMEC-hTERT). Negative controls used to adjust compensation settings are shown (left). ALDH1-positive cells show higher EGF binding and a higher number of EGF receptors (right). DEAB, diethylaminobenzaldehyde. (B) Binding of EGF in ALDH1-negative or ALDH1-positive MECs. Cells were incubated at 4°C with the indicated amounts of EGF and labeled with ALDH1 reagent. Inset: Scatchard analysis of EGF binding. Kd values were 0.32 nM for both the ALDH1+ and ALDH1- fractions. The intensity of the red fluorescence was measured using the channel for red fluorescence (Discosoma Red - ds-red) of the flow cytometer. (C) EGF binding and uptake. Cells were incubated with 10 ng/ml Rh-EGF at 4°C, free Rh-EGF was removed and MECs were counterstained with ALDH1 reagent. To assess binding and uptake, cells were washed with phosphate-buffered saline (PBS) (solid symbols). To assess solely uptake, bound EGF was removed using an acetic acid wash (bordered symbols). (D) Immunofluorescence of EGFR and ALDH1 in aysynchronously growing MECs (human MEC-human telomerase reverse transcriptase).