Millicell ez 4 well glass slides

Vero E6 (2x10⁴ cells/well), A549-ACE2 (2x10⁴ cells/well), or Calu-3 cells (4x10⁴ cells/well) were seeded overnight in Millicell EZ 4-well glass slides (Millipore). Following cytokine treatment and viral infection, cells were washed twice with 1X PBS and fixed for 30 min with 4% PFA at room temperature. In some experiments, cells were stained with DAF-FM diacetate (as described above) before fixation. Next, cells were washed again, blocked with 5% normal goat serum (Vector Laboratories) in 1X PBS containing 0.05% saponin (Sigma-Aldrich) for 20 minutes, and then immunolabeled with indicated primary antibodies for 1 h at room temperature. Following washing, cells were stained with secondary antibodies for 1 h in the dark, washed, and mounted with ProLong Gold Antifade with, or without DAPI (Invitrogen), where NucBlue dye was used instead. Cells were examined using a Leica TCS SP8 Digital LightSheet Laser Scanning Confocal Microscope at the Advanced Light Microscopy and Spectroscopy Laboratory, California NanoSystems Institute at UCLA. Image acquisition was carried out with the CS2 63x or 100x/1.4 oil objectives controlled by Leica Microsystems Application Suite X software.

Eight thousand cells per well were plated in Millicell EZ 4-well glass slides (Millipore, Burlington, MA, USA). Eighteen hours before treatments, the medium was changed for phenol red-free DMEM without fetal bovine serum and cells were incubated at 37 °C under a 95% air, 5% CO₂ atmosphere. After the treatments, cells were fixed for 20 min in 4% paraformaldehyde solution at 37 °C and permeabilized with 100% methanol for 6 min at −4 °C. Next, fixed cells were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature and incubated at 4 °C for 24 h with 1µg/mL of rabbit anti-pS400PR (ab60954, Abcam, Cambridge, UK) in 0.5% bovine serum albumin in PBS. The samples were rinsed thrice in PBS for 5 min each and incubated in the dark with 0.5 µg/mL anti-mouse Alexa Fluor 488-labeled secondary antibodies (A11034, Invitrogen, Carlsbad, CA, USA) for 45 min. Nuclei were stained with 1 µg/mL Hoechst 33342 solution (Thermo Scientific, Waltham, MA, USA). The cells were coverslipped with a fluorescence mounting medium (Biocare Medical, Pacheco, CA, USA). The samples were visualized in a Nikon A1R + STORM confocal microscope.
Specific characteristics of the antibodies described in Section 2.2 and Section 2.4 can be consulted in Table S1.