60 widefield oil immersion objective

Cells were plated in a Nunc Lab-Tek II chamber slide (Thermo) at 5 × 10³ cells per well. The next day, cells were transduced with 2×FYVE-mSCAR to label endosomes. For rVSV/SARS-CoV-2_NG-P, 48 h postransduction, cells were treated with 5 μM E64d (Sigma-Aldrich; catalog no. E8640-250UG) for 30 min, followed by inoculation with rVSV/SARS-CoV-2_NG-P at an MOI of 2. Sixty minutes postinfection, cells were washed 3× with PBS and fixed in 4% PFA. Alternatively, unlabeled cells were treated with pHrodo Red dextran and infected with rVSV/SARS-CoV-2_NG-P for 60 min. Sixty minutes postinfection, cells were washed 3× with PBS and imaged in live cell imaging solution. For cells with 2×FYVE-labeled endosomes, images were acquired on a DeltaVision OMX SR imaging system using a ×60 widefield oil immersion objective (Olympus) with an exposure time of 50 ms, 10% transmission for the AF-488 channel, an exposure time of 100 ms, 10% transmission for the A568 channel, and an exposure time of 150 ms, 10% transmission for the DAPI channel. For cells with dextran Red-labeled endosomes, images were acquired on a DeltaVision OMX SR imaging system using a ×60 widefield oil immersion objective (Olympus) with an exposure time of 50 ms, 10% transmission for the AF-488 channel, an exposure time of 50 ms, 10% transmission for the A568 channel, and an exposure time of 200 ms, 10% transmission for the DAPI channel.

Cells were plated in a Nunc Lab-Tek II chamber slide (Thermo) at 5 × 10³ cells per well. The next day, cells were transduced with 2×FYVE-mSCAR to label endosomes. Forty-eight hours postransduction, cells were treated with pHrodo Green dextran 10,000 molecular weight (MW) (Thermo; catalog no. P35368) at a concentration of 100 μg/mL for 60 min. Alternatively, unlabeled cells were treated with an equal ratio of pHrodo Red dextran 10,000 MW (Thermo; catalog no. P10361) and AF-488 dextran 10,000 MW (Thermo; catalog no. D22910). Thereafter, cells were washed 3× in PBS and placed in live cell imaging solution (Thermo; catalog no. A14291DJ). For 2×FYVE-labeled cells, images were acquired on a DeltaVision OMX SR imaging system using a ×60 widefield oil immersion objective (Olympus) with an exposure time of 50 ms, 5.0% transmission for the AF-488 channel, an exposure time of 50 ms, 10% transmission for the A568 channel, and an exposure time of 150 ms, 10% transmission for the DAPI (4′,6-diamidino-2-phenylindole) channel. For codextran-treated cells, images were acquired on a DeltaVision OMX SR imaging system using a ×60 widefield oil immersion objective (Olympus) with an exposure time of 25 ms, 10.0% transmission for the AF-488 channel, an exposure time of 50 ms, 10% transmission for the A568 channel, and an exposure time of 200 ms, 10% transmission for the DAPI channel.

Cells were plated 2 × 10⁴ cells per well in a Nunc Lab-Tek II chamber slide (Thermo). The next day, intracellular cathepsin L activity was detected using the Magic Red Cathepsin L assay kit (Bio-Rad; catalog no. ICT941). Briefly, cells were incubated in 1× Magic Red and Hoechst 33342 stain for 30 min and then washed 3× with PBS before being placed in live cell imaging solution (Thermo; catalog no. A14291DJ). Images were acquired on a DeltaVision OMX SR imaging system using a ×60 widefield oil immersion objective (Olympus) using an exposure time of 50 ms, 10% transmission for the A568 nm channel, and an exposure time of 100 ms, 10% transmission for the DAPI channel.