4.4. Analysis of Binding to TfR on Mouse Brain Endothelial Cells Using Flow Cytometry

The monoclonal anti-mouse TfR antibody 8D3 was purchased from Novus Biologicals (Novus Biologicals LLC, Centennial, CO, USA). The bEnd.3 mouse brain endothelial cell line (ATCC) was cultured in Dulbecco’s Modified Eagle’s Medium (Merck KGaA) complemented with 10% fetal bovine serum. The SKOV-3 cell line (ATCC) was cultured according to the manufacturer’s protocol. At approximately 80% confluency, cells were harvested using TrypLE^TM Express (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Around 100,000 cells were resuspended in the respective protein solution in PBS + 1% bovine serum albumin (BSA; PBSB) and incubated for 45 min at 4 °C under constant agitation. The supernatant was discarded by centrifugation at 1700 rpm and 4 °C for 4 min. Cells were subsequently resuspended in 100 nM HSA-Alexa Fluor 647 conjugate (produced in-house) in PBSB and incubated for 20 min at 4 °C under constant agitation. The supernatant was discarded as described above and the cells were resuspended in 400 uL ice-cold PBSB. The cells were analyzed using a Gallios^TM flow cytometer (Beckman Coulter Inc., Indianapolis, IN, USA). Gates based on forward and side scatter intensities were used to analyze intact and single cells (Supplementary Figure S3A). All flow cytometry experiments were carried out in duplicates on separate biological samples.