Quantification of the number and size of lysosomes in uninfected and reovirus-infected cells . HBMECs were adsorbed with reovirus T3D at an MOI of 1 PFU/cell. At 18 hpi, cells were fixed and processed for immunofluorescence and confocal microscopy. (A) Representative immunofluorescence images of cells labeled with antibodies specific for LAMP-1 (red) in uninfected and reovirus-infected cells. Nuclei are labeled with DAPI (blue). Lysosome number and size were quantified using ImageJ-Fiji software. Segmented images of confocal data with lysosome areas labeled with LAMP-1 are shown (arrows). Scale bars, 10 µm. (B) Quantification of total, individual, and aggregated LAMP-1–positive lysosomes per cell in uninfected and reovirus-infected cells. An area of <0.78 µm2 corresponds to individual lysosomes; areas of ≥0.78 µm2 are defined as aggregated lysosomes. The results are presented as mean ± SEM. Each data point represents the number of total, individual, and aggregated lysosomes, respectively, in a cell (n = 52 cells pooled from three independent experiments; *, P < 0.05; **, P < 0.01, unpaired two-tailed Student’s t test). (C) Quantification of individual and aggregated lysosome size in uninfected and reovirus-infected cells. Each data point represents the lysosome size quantified with ImageJ-Fiji software (n = 52 cells pooled from three independent experiments; **, P < 0.01; ****, P < 0.0001, unpaired two-tailed Student’s t test).
