Transferrin Receptor Recycling in Fibroblasts

The transferrin receptor recycling protocol was adapted from the literature [49 (link), 76 (link)]. Fibroblasts were plated on glass coverslips 48 h before the experiment. After rinsing with PBS, fibroblasts were kept at 4 °C for 30 min in serum-free DMEM/GlutaMax (Gibco, Thermofisher Scientific). Fibroblasts were then rinsed on ice and incubated in DMEM / GlutaMax / SVF 10% / penicillin-streptomycin 1% supplemented with transferrin (100 μg / mL, T13342, Thermofisher Scientific) at 37 °C for 15 min. Cells were rinsed on ice and fixed in PFA 4% for 10 min. Cells were then immunolabelled against the surface transferrin receptor, therefore with an immunolabelling protocol without permeabilization. Apart from permeabilization, the staining protocol is identical to that presented above. Cells incubated 1 h in primary antibody anti-CD71 (monoclonal rabbit, 13,208, Cell Signaling, 1/300) and 1 h in secondary antibody anti-mouse Alexa Fluor 647 (donkey, A^− 31,571, ThermoFisher Scientific, 1/1000) were then observed under a Leica TCS SP8 AOBS confocal microscope with an oil immersion objective 63x / NA = 1.40 on the ICMQuant platform.