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. 2021 Jul 22;11:14961. doi: 10.1038/s41598-021-94095-7

Figure 4.

Figure 4

Immunofluorescence staining and quantification of IAV-infected PREDICT96-ALI airway tissue. (A) Staining of the nucleoprotein (NP, green) for IAV, actin (phalloidin, grey), β-tubulin (red) and nucleic acids (DAPI, blue), including merged panels for all four stains, within PREDICT96-ALI airway tissue developed using freshly harvested epithelial cells from donor D at 48 h p.i., after 5 weeks ALI culture and following inoculation with A/California/04/09 H1N1 (MOI 0 [mock control], 0.1 or 1). Strong expression of IAV-NP is seen at 48 h p.i. at both MOI 0.1 and 1; noted here is that this is one field of view (FOV), and that there is more cytopathic effect at MOI = 1. Images captured are ×40, z-stacks, slice 25 of 48. Scale bar 50 µm. (B) Quantification of the mean fluorescence intensity (MFI) of the IAV-NP stain (green) or Alexa Fluor 488 signal, indicating significant increase in IAV replication at both 0.1 and 1 MOIs. N = 2 independent experiments with data presented from donor D as representative. Per experiment, N = 3 tissue replicates per donor, time-point and condition (MOI, anti-viral dose). One FOV was analyzed at N = 7 slices (at 5 slice intervals) for each condition. Statistical significance: **p ≤ 0.01 and ***p ≤ 0.001.