Opera phenix high content screening microscope

K562 cells (non-treated or transduced with lentiviral plasmids) were transferred to ice, fixed with 3% paraformaldehyde for 15 min on ice followed by 15 min at room temperature. After three washes with PBS, cells were immunostained in suspension using an adapted version of a protocol described for adherent cells (Mamińska et al., 2016 (link)). Stained cells were resuspended in 0.5% low-melting agarose (Sigma-Aldrich) and transferred to microscopy 96-well plates (Greiner Bio-One). The plates were scanned using Opera Phenix high content screening microscope (PerkinElmer) with 40 × 1.1 NA water immersion objective. Harmony 4.9 software (PerkinElmer) was used for image acquisition and their quantitative analysis. To quantify chosen parameters in the obtained images (number of vesicular structures per cell, mean fluorescence intensity per structure, integral fluorescence intensity per cell and mean pixel intensity), more than 40 microscopic fields were analyzed per each experimental condition. Maximum intensity projection images were obtained from 7 to 8 z-stack planes with 1 μm interval. Pictures were assembled in Photoshop (Adobe) with only linear adjustments of contrast and brightness.

Infectivity assays were performed as in the accompanying manuscript (see Zeng et al. [31 (link)]). Briefly, VERO E6 cells were grown in 96 well imaging plates and transfected with individual gapmers at 0.5 µM using Lipofectamine 2000 (Thermofisher). Six hours post transfection, the media was replaced and infected with SARS-CoV-2 at an MOI of 0.5 PFU/cell and, simultaneously, a titration of NSC95397 was added to different wells with or without the addition of 0.5 µM remdesivir. Twenty-two hours post-infection, cells were fixed, permeabilised and stained for SARS-CoV-2 N-protein using Alexa488-labelled-CR3009 antibody [64 (link)] and nuclei using DRAQ5 (647 nm wavelength). Imaging was performed using a 5× lens in an Opera Phenix high content screening microscope (PerkinElmer) equipped with the Harmony software. The whole well area was delineated, and area of Alexa488/N protein and DRAQ5/DNA signals were determined for each well. The Alexa488/N intensities were normalised to DRAQ5/DNA and to vehicle treated samples.