Alexa fluor 647 conjugated transferrin

Cells were incubated with Alexa Fluor 647-conjugated transferrin (T23366, Life Technologies, distributed by ThermoFisher Scientific, Waltham, USA) for 15 min, washed with PBS and fixed with paraformaldehyde (PFA). Images were acquired on a confocal microscope (Nikon Eclipse T1, Nikon Tokyo, Japan) with spinning disk (3I, Denver, CO, USA) with a 100x oil objective (Nikon CFI Apo TIRF 100x NA 1.4). z-stack images in a total range of 10 µm were acquired and maximum projections obtained with ImageJ. The quantification of the fluorescence signal was performed in ImageJ by measuring the mean grey value for the transferrin channel (far red) per pixel in the area of the cells.

hTERT-RPE-1 cells grown to confluence in eight-chamber cell culture slides and serum-starved for 24 h were cooled on ice for 10 min and the culture medium was switched to ice-cold serum-free medium for 5 min to block endocytosis. Alexa Fluor647-conjugated transferrin (Life Technologies, catalog no. T2366) was diluted in serum-free culture medium to a final concentration of 0.25 mg/mL and was prewarmed to 37 °C before adding to the cells. Cells were incubated with medium containing Alexa Fluor647-transferrin at 37 °C for 30 min, washed thrice with serum-free medium, fixed in 4% PFA for 15 min, washed and costained for ADAMTS9 and acetylated α-tubulin and imaged using super-resolution confocal microscopy at ×1000 magnification.

Alexa Fluor 647-conjugated transferrin (Invitrogen, T23366) was used to track CME and Alexa Fluor 647-conjugated 10,000 MW dextran (Invitrogen, D22914) was used as a marker for fluid-phase uptake during macropinocytosis. To study reovirus co-transport with cargoes taken up by distinct endocytic pathways, CNs were incubated with 3×10¹⁰ particles of Alexa Fluor 488-labeled T3SA+ reovirus and fluorescent transferrin or dextran at final concentrations of 50 μg/ml and 100 μg/ml, respectively, in culture medium. Cells were incubated at 37°C for 30 min, washed twice with HBSS, returned to culture medium, and imaged immediately under live-cell imaging conditions. Multichannel time-lapse images were acquired simultaneously using HyD detectors. Maximal intensity projections of time-lapse images were used to visualize trajectories of transported reovirus particles. Kymographs were generated along individual trajectories using Fiji ImageJ and cotransport of reovirus with transferrin or dextran was determined from the overlap of corresponding fluorescence signals on kymographs.