Computational protocol: An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

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[…] Cells were fixed with 4% PFA and washed and blocked with blocking buffer (PBS, 1% bovine serum albumin, and 0.1% saponin). Primary and secondary antibodies were diluted in the blocking buffer and incubated for 1 h. Nuclei were stained with Hoechst 33342 during the incubation with the secondary antibody for automated image acquisition. Alternatively, nuclei were stained with DAPI for confocal microscopy. Confocal microscopy was performed at room temperature using a laser-scanning microscope 700 (ZEISS) with a 63× 1.4 DIC Plan-Apochromat oil-immersion objective. ZEN digital imaging software (ZEISS) was used for image acquisition and processing of the images. All images were exported as TIFF images, and figures were finalized in Adobe Illustrator (Adobe). [...] A Cellomics Arrayscan VTI HCS Reader (Thermo Fisher Scientific) was used to acquire cell images in the Hoechst, FITC and TRITC filters using either the 20×, 10×, or 5× lens for automated fluorescence signal acquisition.A minimum of 400 cells per experiment was analyzed using the Cellomics SpotDetector V3 algorithm to determine the mean number of p62 and LC3 puncta per cell as well as the mean number of LC3 puncta area per cell. The Hoechst channel was used to set the autofocus; LC3 puncta were detected using the FITC filter, and p62 puncta were detected using the TRITC filter. An equal fixed exposure time was automatically set for all the samples, and LC3 and p62 puncta were detected in a cell area that excluded the nucleus. The numbers of nuclei (valid object count), LC3 and p62 puncta per cell (spot per cell), and LC3 and p62 puncta area per cell (spot area per cell) were counted using this approach. To assess cell infection rate, cells were stained with either anti-capsid antibody when infected with EMCV or with anti-VP1 or anti-2bc antibodies, when infected with CV, before to image 5–30 fields of cells per sample using the Hoechst, FITC, or TRITC filters. The percentage of EMCV- or CV-positive cells was determined by analyzing the collected images using the cell counter application in ImageJ. […]

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