Influenza Virus Infection Assay in MDCK Cells

Monolayers of MDCK cells (6-well plate format, 1 × 10⁶ cells/well) were infected with PR8/WT, PR8/AA and PR8/Len for 1 h at RT, overlaid with agar, and incubated at 33 °C, 37 °C, or 39 °C [13 (link)]. At three days p.i., cells were fixed for 1 h at RT with 4% paraformaldehyde (PFA) and the overlays were removed. Cells were then permeabilized (0.5% Triton X-100 in PBS) for 15 min at RT and immunostained using the anti-NP mAb HB-65 and Vector kits (Vectastain ABC Vector kits and DAB HRP substrate kit; Vector) according to the manufacturer’s specifications.

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Rodriguez L., Blanco-Lobo P., Reilly E.C., Maehigashi T., Nogales A., Smith A., Topham D.J., Dewhurst S., Kim B, & Martínez-Sobrido L. (2019). Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines. Viruses, 11(10), 928.

Publication 2019

Vectastain ABC Vector kits DAB HRP substrate kit

Agar Cells Mdck cells Paraformaldehyde Triton x 100 Vector

Corresponding Organization : University of Rochester

Other organizations : Emory University, Kyung Hee University

Top 5 similar protocols

Variable analysis

independent variables

Virus strain (PR8/WT, PR8/AA, PR8/Len)

dependent variables

Viral plaque formation

control variables

Cell line (MDCK cells)
Cell density (1 × 10^6 cells/well)
Incubation temperature (33 °C, 37 °C, 39 °C)
Infection duration (1 h)
Room temperature (RT)
Fixation duration (1 h)
Permeabilization duration (15 min)
Immunostaining method (anti-NP mAb HB-65, Vectastain ABC Vector kits, DAB HRP substrate kit)

controls

No positive or negative controls are explicitly mentioned.

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