Zombie nir live dead dye

For flow cytometry analysis, adherent cells were recovered by trypsinization and washed in PBS with 2 mM EDTA (PBS/EDTA). Cells were stained with fixable Zombie NIR Live/Dead dye (BioLegend) for 6 min at room temperature. Excess stain was quenched with FBS-complemented DMEM. Cells were fixed in 4% paraformaldehyde before intracellular staining.
For intracellular detection of SARS-CoV-2 nucleoprotein, cells were permeabilized for 15 min with Intracellular Staining Perm Wash Buffer (BioLegend). Cells were then incubated with 1 μg/ml CR3009 SARS-CoV-2 cross-reactive antibody (a kind gift from Dr. Laura McCoy) in permeabilization buffer for 30 min at room temperature, washed once and incubated with secondary Alexa Fluor 488–Donkey-anti-Human IgG (Jackson Labs). All samples were acquired and analyzed on a NovoCyte 3005 Flow Cytometer System (Agilent).

Animals were euthanized and the spleen and mesenteric lymph nodes were collected in ice-cold PBS. Tissue was crushed on top of a 70 μm cell strainer with a plunger from a 5 ml syringe and washed through using ice-cold PBS. Cells were spun down at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the pellet resuspended in FCS:DMSO (9:1, v/v). Cells were stored in cryovials in liquid N₂ until the day of analysis. Before analysis, cells were thawed, washed in ice-cold FACS buffer (PBS with 2% FCS), resuspended in FACS buffer on ice and counted using a Vi-CELL Counter (Beckman Coulter). Maximum 10⁶ cells were allocated per well in 200 μl FACS buffer. Cells were washed once, spun down at 2000 rpm for two minutes and stained with 50 μl antibody cocktail. Antibodies were diluted 1:200 and are listed in the Key Resources Table. Zombie NIR Live/Dead Dye (Biolegend) was used to stain for live cells. Cells were stained for 30 minutes at 4°C and washed twice in FACS Buffer at 2000 rpm for two minutes at 4°C. Samples were kept on ice until acquisition on a BD LSRFortessa analyzer (BD Biosciences). Data were analyzed using FlowJo 10.

Cells were washed with 1xPBS for staining. For Live/Dead staining cells were stained with Zombie NIR Live/Dead Dye (Biolegend) at 1:1000 for 20 min at room temperature and the washed once with FACS Buffer. Fc receptor blocking step was performed using HuTrustain FcX (Biolegend) at 1:100 for 10 min at room temperature protected from the light and then washed once with FACS Buffer. For surface staining, cells were incubated for 30 min at 4 °C with a mix of respective conjugated antibodies. Immediately following staining, cells were once washed with FACS buffer and the samples were analyzed using BD LSR Fortessa. Gating strategies for individual experiments are shown in supplementary Supplementary Fig. 7.