Cellsens standard version 2

Endocytosis measurements were carried out using FM1-43FX (ThermoFisher Scientific) as described previously^{17 (link)}. Briefly, HIE monolayers were treated with 10 μg/ml of FM1-43FX for 10 min at 37 °C with either VLP alone or VLP preincubated with M4 for 1 h at 37 °C. Monolayers were washed with prechilled PBS and fixed in 4% PFA for 20 min. followed by nuclei staining with 300 nM DAPI for 5 min at room temperature. Endocytic trafficking was measured by the presence of fluorescent puncta which were observed by epifluorescence microscopy using Olympus cellSens Standard Version 2.3 software. Quantitation of the number of fluorescent puncta was done using J-image. Every experiment was repeated at least three times with 4 images analyzed per condition in each experiment.

Differentiated J2 HIE monolayers (incubated with virus/VLPs for 10 min; bafilomycin for 1 h) were incubated with 50 nM LysoTracker (ThermoFisher Scientific) for 10 min. The cells were washed twice with CMGF(-) and LysoTracker staining of acidic compartments in treated and untreated J2 HIEs (mock) was observed by epifluorescence microscopy using Olympus cellSens Standard Version 2.3 software. Endocytosis measurements were carried out using FM1-43FX (ThermoFisher Scientific). HIE monolayers were treated media containing 10 μg/mL of FM1-43FX with or without VLPs for 10 min at 37 °C. Endocytosis was stopped by washing with prechilled PBS and the cells were fixed in 4% PFA for 20 min and nuclei were stained with 300 nM DAPI for 5 min at room temperature. Quantitation of the fluorescence was done using J-image. Briefly, every experiment was repeated at least three times with 3–4 images analyzed per condition in each experiment. Identical elliptical regions-of-interests (ROIs) were drawn per field and mean fluorescence intensity from these ROIs was measured.