Columbus system

Neutralizing activities of antibodies against WT SARS-CoV, WT SARS-CoV-2, WT SARS-CoV-2 VOCs, and SARS-CoV-2 bearing different single-point mutations were quantified based on rVSVs, as previously described (33 (link)). A series of diluted monoclonal antibodies was mixed with pseudoviruses carrying gene encoding for the S protein of SARS-CoV, SARS-CoV-2, or its variants, and incubated at 37 °C for 1 h, respectively. The mixture was then transferred to BHK21-hACE2 cells seeded in a 96-well microplate and incubated for 12 h. Fluorescence images were captured by Opera Phenix (PerkinElmer) and quantitatively analyzed by the Columbus system (PerkinElmer). The percentage reduction of EGFP in each well as compared with the control wells was calculated. IC₅₀ values were determined by the four-parameter logistic regression using GraphPad Prism (version 8.0.1).

The cytotoxicity assay was performed using an xCELLigence real-time cell analyzer (RTCA) System (ACEA Biosciences, San Diego). Impedance-based RTCA was used for label-free and real-time monitoring of cytolysis activity. The cell index (CI) based on the measured cell-electrode impedance was used to measure cell viability. Cytotoxicity was calculated via the following formula: ((CI (target cells only) – CI (target cells + T cells))/CI target cells only) × 100%. S-293T cells were seeded at a density of 1 × 10⁴ cells per well and grown for 24 hours. Control T or SARS-CoV-2-S CAR-T cells with or without drug treatment were then added to the RTCA unit at different ratios (T cells: S-293T cells = 1:1, 3:1, 6:1 or 10:1 or 5:1). The impedance signals were recorded for 24–96 hours at 5-min intervals.
For GFP fluorescence detection, eGFP-S-293T cells were seeded at a density of 2 × 10⁴ cells per well. CTL T or SARS-CoV-2-S CAR-T cells were then added to eGFP-S-293T cells at different ratios (T cells: eGFP-S-293T cells = 1:1, 3:1, 6:1 or 10:1). Fluorescence images were obtained by Operetta CLS equipment (Perkin-Elmer, Waltham) at 96 hours. For quantitative determination, fluorescence images were analyzed, and the numbers of GFP-positive cells were counted by a Columbus system (Perkin-Elmer, Waltham).

U-2-OS cells were grown on clear bottom 96-well plates (Greiner) and either pre-extracted with cold 0.5% Triton X-100 CSK buffer for 5 min before fixation or directly fixed in 4% formaldehyde for 10 min. For EdU (5-ethynyl-2′-deoxyuridine) labeling, cells were incubated with 40 μM EdU for 20–30 min. EdU was detected using Click-iT Plus EdU Kit for Imaging (C10640). Plates were imaged on a Perkin Elmer Operetta high-content imaging system or Olympus Scan-R imaging system using a 20x objective. 35 fields per well were imaged, and ∼2,000 cells per condition were analyzed. Single-cell fluorophore intensities were extracted using the Columbus system (Perkin Elmer) or Scan-R analysis software. Cell cycle phases were gated based on DAPI and EdU or PCNA intensities. Graphs were generated using Tableau 2019.3 and GraphPad Prism.9 software.

Changes in cell morphology following drug treatments were assessed by seeding cancer cells into a 96-well plate at a density of 6 × 10³ cells/well and culturing for 24 h. The cells were treated for 12 h with free dihydroartemisinin (10 μM), free epirubicin (5 μM), free dihydroartemisinin plus free epirubicin (10 μM dihydroartemisinin, 5 μM epirubicin), dihydroartemisinin liposomes (10 μM), epirubicin liposomes (5 μM), or dihydroartemisinin plus epirubicin liposomes (10 μM dihydroartemisinin, 5 μM epirubicin). Culture medium was used as a blank control. After incubation, cells were harvested and washed twice with cold PBS (pH 7.4), and mitochondria were stained with Mitotracker Deep Red (20 nM, Life Technologies Corporation, Carlsbad, NM, USA) at 37 °C for 30 min. Afterwards, nuclei were stained with Hoechst 33342 (5 μg/mL) for 10 min. Finally, cells were imaged using the Operetta High-Content Screening System (Perkin Elmer, Waltham, MA, USA), and calculations were performed using the Columbus system (Waltham, MA, USA).

Imaging of each full section was done with the Opera Phenix™ high-content screening system and Harmony software (Perkin Elmer, Montreal, QC, Canada) with a field overlap of 5%, at a 40× magnification. The images were further analyzed using Columbus™ system (Perkin Elmer, Montreal, QC, Canada) to quantify the signal of the immunofluorescent staining. Analyses were applied which recognized DAPI as a nucleus marker and SRC as a cytoplasmic marker to segment the cells for quantification of fluorescence intensity in each cellular compartment. The 4-HNE and 8-OHG intensities were used to determine oxidative damage in membranes and DNA, respectively. Analyses were done separately in the caput, corpus and cauda epididymides, as well as in the epithelium and in the interstitial cells (endothelial, smooth muscle, and conjunctive cells).

Pseudovirus-based neutralization assays were performed as previously reported (Xiong et al., 2020 (link)). BHK21-hACE2 cells were pre-seeded in 96-well plates. Serially diluted (3-fold gradient) serum samples were mixed with diluted VSV-SARS-CoV-2-Sdel18 virus and incubated at 37°C for 1 h. The mixture was added to the pre-seeded BHK21-hACE2 cells. After 12 h of incubation, fluorescence images were obtained with Opera Phenix or Operetta CLS equipment (PerkinElmer). For quantitative determination, fluorescence images were analyzed by the Columbus system (PerkinElmer), and the numbers of GFP-positive cells for each well were counted to represent infection performance. The neutralization titer for each sample was expressed as the maximum dilution fold (ID₅₀) required to achieve infection inhibition by 50% (50% reduction in GFP-positive cell numbers as compared with controls).