Computational protocol: Generation of Unique Poliovirus RNA Replication Organelles

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Protocol publication

[…] H1-HeLa cells were grown on glass coverslips in a 24-well TC plate. For all experiments, approximately 1.5 × 105 cells were infected at an MOI of 50 PFU/cell. Following infection, cells were washed three times with phosphate-buffered saline (PBS) and permeabilized with −20°C methanol at 4°C for 10 min. Cells were then washed three times with PBS and incubated overnight with blocking buffer (20% goat serum, 0.05% saponin in PBS) at 4°C. Cells were then incubated with the primary antibody for 3 h at room temperature. All primary antibodies were diluted in blocking buffer. Cells were washed three times with PBS and then incubated with the appropriate secondary antibody for 1 h at room temperature. All secondary antibodies were diluted in blocking buffer. Cells were next washed three times with PBS and mounted using ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen). For all images, a Z-stack of approximately 30 slices was taken. A deconvolved maximum projection was generated using the AutoQuant X3 software (MediaCybernetics). Deconvolution was performed using AutoQuant X3’s three-dimensional blind (adaptive point spread functional [PSF]) deconvolution algorithm. Colocalization analysis was performed by ImageJ software using the Manders coefficient plug in. Rr values represent the Pearson’s correlation coefficient. Correlation coefficients were calculated for two or three random fields of multiple cells, and the average values are displayed in the figures. For colocalization of dsRNA with GFP-tagged proteins, correlation coefficients were determined for three images containing only transfected cells. […]

Pipeline specifications

Software tools AutoQuant, ImageJ
Application Microscopic phenotype analysis
Organisms Enterovirus C, Homo sapiens
Diseases Infection, Picornaviridae Infections