Live cell imaging dishes

To perform single-particle tracking analysis of Rab7-positive or Arl8b-positive endosomes, HeLa^ORF3a-Strep cells seeded on glass-bottom tissue culture-treated live-cell imaging dishes (ibidi) were either left untreated or Dox-treated (1 µg/mL) for 8 h in complete DMEM medium in a cell culture incubator. After 8 h, the cells were co-transfected with GFP-Rab7 and Arl8b-tomato expressing plasmids and incubated further for 16 h in complete DMEM with or without Dox (1 µg/mL). Before the start of the time-lapse confocal imaging, cells were washed with 1X PBS, phenol red-free DMEM media (Gibco) supplemented with 10% FBS was added, and live-cell imaging was performed using a ZEISS LSM 980 Elyra 7 super-resolution microscope with a 63×/1.4 NA oil immersion objective. To measure the mobile fraction and average speed of Rab7- or Arl8b-positive endosomes from time-lapse images, the “TrackMate” plugin of Fiji software was used with the following parameters:
•Vesicle diameter, 1 µm
•Detector, DoG
•Initial thresholding, none
•Tracker, Simple LAP tracker
•Linking max distance, 2 μm
•Gap-closing max distance, 2 μm
•Gap-closing max frame gap, 2
•Filters, none
After imaging, all the data were exported to a Microsoft Excel spreadsheet (2016) for further analysis.

The RUSH assay was performed as previously described^{25 ,63 (link)}. Briefly, HeLa^ORF3a-Strep cells seeded on glass-bottom tissue culture-treated live-cell imaging dishes (ibidi) were either left untreated or Dox-treated (1 µg/mL) for 8 h in complete DMEM in a cell culture incubator. After 8 h, cells were co-transfected with Str-KDEL-IRES-SBP-mCherry-CtsZ (Cathepsin Z reporter) and LAMP1-GFP expressing plasmids and incubated for 16 h in complete DMEM with or without Dox (1 µg/mL). Before the start of the experiment, the cells were washed with 1X PBS, and phenol red-free DMEM (Gibco) supplemented with 10% FBS was added. Using a ZEISS 710 confocal microscope with a plan apochromat 63×/1.4 NA oil immersion objective, single-plane confocal images were acquired before and after the addition of biotin (final concentration 40 µM; Sigma-Aldrich), which causes a synchronous release of the reporter from the hook, for the indicated time points. Pearson’s correlation coefficient between mCherry-CtsZ and LAMP1-GFP was quantified using the “JACoP” plugin of the Fiji software.