Alexa fluor 633 transferrin

Endosomal pH was measured by a ratiometric method as described previously (Kondapalli et al., 2015 (link)). Cells were starved in serum-free media for 30 min to remove residual transferrin, and incubated with pH-sensitive FITC-transferrin (Thermo Fisher Scientific) (75 μg/mL) and pH insensitive Alexa Fluor 633-transferrin (Thermo Fisher Scientific) (25 μg/mL) at 37°C for 1 h. Transferrin uptake was stopped by placing cells on ice. Cells were then washed twice with ice-cold PBS pH 7.4 to remove excess transferrin, followed by ice-cold PBS pH 5.0 and pH 7.4 to remove surface bound transferrin. Cells were trypsinized and analyzed by flow cytometry. Cells with both FITC-transferrin and Alexa Fluor 633-transferrin were gated and a ratiometric analysis of ~10,000 cells for each biological replicate was used to determine the endosomal pH. A standard curve with different pH values 4.5, 5.5, 6.5, and 7.5 was constructed using the Intracellular pH Calibration Buffer kit (Thermo Fisher Scientific) in the presence of 10 μM K⁺/H⁺ ionophore nigericin and 10 μM K⁺ ionophore valinomycin to equilibrium the endosomal pH with the calibration buffers.