Figure 6. Viral N staining distinguishes two subpopulations of SARS-CoV-2 infected Vero E6 cells.

(A) Vero E6 cells were exposed to a low virus input of SARS-CoV-2 (20A.EU2 variant; MOI = 0.28). At day 2 p.i., cells were collected, fixed, permeabilized and stained with an antibody that binds to the viral nucleoprotein N. Dot plots show N expression in noninfected (lower left quadrant) and infected (lower right quadrant) cells from a representative experiment with multiple individual samples. Single cell analysis was done on numerous independent cells, and the individual plots shown in the figure are based on the analysis of 8,000 – 10,000 Vero E6 cells by flow cytometry. The graph shows two distinct subpopulations of infected cells, i.e., ‘N low’ (marked by a red line) and ‘N high’ (marked by a blue line). (B) Vero E6 cells were exposed to a high virus input of SARS-CoV-2 (20A.EU2 variant 1:10; MOI = 28), without (top panels) or with 10 μg/ml of spike-neutralizing antibody R001 (bottom panels). At 4 h p.i., half of the samples were washed to remove virus input and were administered fresh medium (without or with R001) and incubated for another 20 h followed by N staining. The other half of the cells were collected at 4 h p.i., fixed, permeabilized and stained with an antibody that binds to the viral N protein. Dot plots show N expression in infected cells from a representative experiment with multiple individual samples. Single cell analysis was done on numerous independent cells, and the individual plots shown in the figure are based on the analysis of 8,000 – 10,000 Vero E6 cells by flow cytometry. The two distinct subpopulations of infected cells, i.e., ‘N low’ (marked in red) and ‘N high’ (marked in blue) are indicated. The numbers refer to the distribution of the cells (i.e., percentage of total cell population). The dot plot in black (left panel) represents the noninfected cell control. To determine non-specific background of the spike-neutralizing antibody R001, in the same experiment non-infected controls were also incubated with R001 and stained with the fluorescently labeled secondary anti-rabbit antibody. As those dot-plot panels were identical to the non-infected control sample (without R001), they are not presented in the figure for clarity of the figure.

FSC: Forward scatter; MFI: Mean fluorescence intensity; p.i.: Postinfection.