Astrocytes or HEK293 cells were rinsed and incubated in serum-free medium for 30 minutes to remove any residual transferrin and then were exposed to 100µg/ml transferrin conjugated with Alexa Fluor 568 or 633 (Invitrogen) at 37° C for 55 minutes. Uptake was stopped by chilling the cells on ice. External transferrin was removed by washing with ice-cold serum-free DMEM and PBS, whereas bound transferrin was removed by an acid wash in PBS at pH 5.0 followed by a wash with PBS at pH 7.0. Surface bound transferrin (less than 5% of total) was determined with a parallel sample incubated on ice and used for background subtraction. The fluorescence intensity of internalized transferrin was measured for at least 5,000 cells by flow cytometry using the FACSAria (BD Biosciences, San Jose, CA) instrument and the average intensity of the cells population was recorded.
