Operetta high content imaging system

MCF7 human breast cancer cells were plated at 5×10³ cells/well in black polystyrene glass-bottomed 96 well plates and treated the next day with 0.2 μg/mL doxorubicin plus indicated LIMK inhibitor concentrations for 18 hours. Cells were washed, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (v/v) and blocked for 1 hour with 1% BSA (w/v). Fixed cells were stained with rabbit antibody against phospho-Ser3 cofilin, then Alexa 488 anti-rabbit antibody (Invitrogen), Texas red Phalloidin (Molecular Probes) and DAPI (Sigma). After washing, cells were imaged on a High Content Imaging Operetta system (PerkinElmer) and phospho-Ser3 cofilin fluorescence intensity for each cell quantified using Harmony^® High Content Imaging and Analysis Software (PerkinElmer), and plotted as percent change from DMSO alone-treated control for eight independent replicate determinations. EC₅₀ values were calculated from dose-response curves using Prism 5 (GraphPad).

The assay is described in detail elsewhere [30 (link)]; briefly, Huh 7-hACE2 cells were seeded overnight to adhere to a 96-well plate, treated with serial dilution of the compounds, and then infected with SARS-CoV-2 with appropriate controls (vehicle, not infected, infected and not treated, and infected and treated with the control inhibitor hydroxychloroquine). The plates were incubated for 20 h at 37 °C, and then fixed with 4% PFA, permeabilized with 0.1% of Triton-X for 15 min and incubated in blocking buffer (PBS containing 1% of BSA).
The antibody mSIP-3022 was diluted in blocking buffer and incubated for 2 h at 37 °C. The cells were washed twice in PBS and incubated with the secondary antibody AlexaFluor488-conjugated goat anti-mouse IgG (Cat No. A-11001, ThermoFisher, Rockford, IL, USA) plus DAPI for 1 h at 37 °C. Each plate was kept in PBS after washing. Digital images were acquired using the Operetta high content imaging system (Perkin Elmer, Walthem, MA, USA). The digital images were taken from nine different fields of each well. The total number of cells (nuclei) and the number of infected cells were analysed using the Columbus Image Data Storage and Analysis System (Perkin Elmer, Waltham, MA, USA).

At the end of each exposure/treatment, H292 cells were trypsinized, counted and seeded in a 96-well (CellCarrier™-96; PerkinElmer #6005550) in triplicate at the density of 1*10³ cells/well and then placed in the incubator (5%CO₂; 37°C) for 24 h. Then cells were labeled for nuclei (NucBlue™ Live cell Stain, Thermo-Fisher Scientific #R37605), membranes (CellMask™ Green Plasma Thermo-Fisher #C37608) and ACE-2 protein receptor (primary antibody: mouse anti-hACE-2; R&D Systems, #MAB933. Secondary antibody Alexa Fluor™ 546 goat anti-mouse IgG; thermo Fisher Scientific, #A11003). Membrane protein expression was assessed by High Content Screening (HCS) analysis using the PerkinElmer Operetta High-Content Imaging System. A dose-response curve and IC50 for ACE-2 expression were calculated, related to both nicotine in the exposed basal media and puff number. Air exposure and incubator controls were used for all the experiments. HCS analysis of ACE-2 protein expression was evaluated following membrane segmentation. Plates were read under confocal conditions using the 20x long WD objective. Enough fields were imaged to capture at least 500 cells/well. All images were analyzed using Harmony high-content imaging and analysis software (PerkinElmer). Final output values from the analysis were expressed as mean fluorescence intensity (MFI) percentage of control per well.

A-673 and SJCRH30 cells were plated into black 96-well cell carrier plates (PerkinElmer) at a density of 3500 cells/well and allowed to adhere overnight. The cells were treated in triplicate with DMSO or the indicated concentrations of ATXII for 6 or 24 h, then fixed with paraformaldehyde. After fixation, the cells were incubated in a blocking solution of 10% bovine calf serum in DPBS for 20 min at room temperature. The cells were then incubated in a primary antibody against γ-H2A.X (1:400; Cell Signaling Technology) diluted in 1% bovine serum albumin/0.3% Triton X-100 in DPBS, overnight, at 4 °C. The cells were subsequently washed with DPBS and incubated with an Alexafluor-594-conjugated secondary antibody (1:1000; Life Technologies) for 1 h at room temperature. The plates were washed with DPBS, and the nuclei stained with NucBlue live cell stain (Life Technologies) diluted in DPBS. Images were collected using an Operetta high-content imaging system (PerkinElmer) using a 20× long working distance objective and analyzed with the Columbus Image Data Storage and Analysis System (PerkinElmer). A minimum of three fields were collected per well, with all concentrations tested in triplicate.