Sorp lsrii analytic flow cytometer

Vero E6 were seeded overnight in 24-well plates at a density of 2x10⁴ cells per well. Following cytokine treatment and viral infection with SARS-CoV-2 (MOI = 0.1), cells were washed and stained with 1 µM DAF-FM diacetate as described above. Next, cells were dissociated with 0.25% Trypsin-EDTA and fixed for 30 min with 4% PFA at room temperature. Vero E6 cells were then washed once with 1X PBS and twice with warm phenol red-free DMEM, and immediately acquired on a SORP LSRII Analytic Flow Cytometer by gating on live, single cells, according to FSC-SSC parameters. FACSDiva software version 8.0.2 was used for acquisition. FlowJo software version 10.7.1 was used to analyze flow cytometry data, which were used to generate the dose-response curves. For uninfected Vero E6 or A549-ACE2 cells, acquisition of live, single cells, was performed immediately after DAF-FM diacetate staining without fixation on either a SORP LSRII Analytic Flow Cytometer or a SORP LSRFortessa X-20 (BD Biosciences). In some experiments, Vero E6 cells were additionally treated with IFN-γ at 800 or 1600 U/mL; IL-1β at 20 or 40 ng/mL; or cytokine combinations of IFN-γ and IL-1β at 800/20 or 1600/40, respectively, and double stained with DAF-FM diacetate and SYTOX Red dead-cell indicator (Invitrogen).

Vero E6 were seeded overnight in 24-well plates at a density of 2x10⁴ cells per well. Following cytokine treatment and viral infection with SARS-CoV-2-mNG (MOI = 0.1), cells were washed, dissociated with 0.25% Trypsin-EDTA (Gibco), and then fixed for 30 min with 4% PFA (Thermo Scientific) at room temperature. Live Vero E6 cells were acquired according to FSC-SSC parameters and doublet exclusion on a SORP LSRII Analytic Flow Cytometer using the FACSDiva software version 8.0.2 (BD Biosciences). FlowJo software version 10.7.1 (BD Biosciences) was used to analyze flow cytometry data, which were used to generate the dose-response curves.