Harmony high content analysis software

nAbs were quantitated against replication-deficient SARS-CoV-2 ancestral (Wuhan) Spike pseudotyped lentivirus particles as previously described⁶⁰ . Briefly, pseudovirus particles were generated by co-transfecting a Spike expression construct pCG1-SARS-2-S Δ18^{61 (link),62 (link)} and GFP-luciferase vector with lentivirus packaging and helper plasmids into HEK293T cells, using Fugene HD (Promega)^{63 (link)}. Pseudovirus particles were incubated with serially diluted serum or BALF samples at 37 °C, 5% CO₂ for 1 h prior to spinoculation (800 x g, 35 °C) of ACE2 over-expressing HEK293T cells. Seventy-two h post-transduction, cells were fixed and stained with Hoechst 33342 (NucBlue™ Live ReadyProbes™ Reagent, Invitrogen) as per the manufacturer’s instructions, and imaged using the Opera Phenix high content screening system (Perkin Elmer). The percentage of GFP positive cells was enumerated (Harmony® high-content analysis software, Perkin Elmer) and neutralising endpoint titre determined as the dilution required for ≥50% inhibition of infection (EC₅₀), estimated by sigmoidal curve and interpolation in GraphPad Prism software.

To quantify neurite damage following Aβ_25–35 treatment (500 nM, 2 µM, 20 µM), neurons were fixed with 4% paraformaldehyde-DPBS at RT for 15 min. Cells were washed with DPBS three times prior to immunostaining. Cells were simultaneously permeabilised and blocked in DPBS containing 0.25% Triton-X and 10% donkey serum (Life Technologies) for 1 h at RT. Chicken anti-MAP2 (Abcam, ab5392, 1:1000) primary antibodies were diluted in DPBS containing 0.25% Triton-X and 5% donkey serum. Cells were washed three times with TBST and incubated for 1 h at RT in secondary antibody goat anti-chicken 647 diluted in blocking solution (Life Technologies, A-21449, 1:500). Cells were then washed once with Hoechst 33342 for 30 s (Thermo Fisher, 0.01 μg/ml diluted in DPBS). Cells were then washed a further three times with DPBS prior to imaging on an Opera Phenix High Content Screening System (Perkin Elmer) under 20 × magnification. Immunofluorescent images were analysed using the Harmony High-Content Analysis Software (Perkin Elmer) using a neurite length assay developed in the analysis interface. First, intact nuclei were identified following positive staining by Hoechst. Neurites protruding from cell bodies stained positive for MAP2 in the 647 channel were then traced and quantified.

JC-1 was dissolved in DMSO as a stocking solution (200 μM). For immunofluorescence assay, MKN45 and NCI-N87 cells were seeded in 96-well black/clear bottom plates (Corning, NY, USA). Add JC-1 (200 μM) to each well to make the final concentration at 2 μM. The cells were incubated with JC-1 for 15 min and washed twice with PBS. Then the cells were observed and images were acquired using Opera Phenix High-Content Screening System (PerkinElmer, Waltham, MA, USA). The ratio of red/green fluorescence was analyzed via Harmony^® high-content analysis software (PerkinElmer).

HT1080 cells were plated on CellCarrier-96 Ultra (Perkin Elmer) at 8 × 10³ cells/well and treated with spautin-1, HCQ, bafilomycin A1, SAR405, or rotenone in the presence or absence of 2DG (10 mM) for 4 h. For siRNA experiments, HT1080 cells were transfected with control siRNAs or combination of USP10 and USP13 siRNAs. After 48 h incubation, the cells were treated with vehicle or spautin-1 (10 μM) under control or 2DG-stressed conditions for 4 h. The cells were subjected to fluorescent immunostaining using anti-ATF4 rabbit monoclonal antibody (Cat#: 11815, Cell Signaling Technology), anti-XBP1s mouse monoclonal antibody (Cat#: 27901, Cell Signaling Technology), Alexa Flour Plus 647 goat anti-rabbit IgG secondary antibody for ATF4 (Cat#: A32733, Thermo Fisher Scientific), Alexa Flour Plus 488 goat anti-mouse IgG secondary antibody for XBP1s (Cat#: A32723, Thermo Fisher Scientific), and nuclear staining with Hoechst 33342, as described previously^{25 (link)}. Fluorescent images (nine fields per well) were acquired using a 20 × water objective lens by Operetta CLS (Perkin Elmer). Quantification of ATF4 and XBP1s mean intensities in nuclei was performed using Harmony high-content analysis software (Version 4.9, Perkin Elmer).

hPSCs were cultured at 30,000 cells/cm² in Matrigel-coated 96-well plates (Perkin Elmer CellCarrier) until reaching 60% confluent before fixing with 4% PFA. Fixed cells were perforated using 0.01% Triton X-100 and 0.05% Tween 20 [(diluted in phosphate-buffered saline (PBS)]. The cells were then incubated with mouse-anti human OCT4 (C-10 clone; No. sc-5279, 1:100; Santa Cruz Biotech) and subsequent secondary antibody using goat-anti mouse Alexa488 (No. A11001, 1:1000; Invitrogen) and counterstained with 0.5 μg/mL DAPI. Immunofluorescence images were captured using Operetta High-Content System (Perkin Elmer) and analyzed using Harmony High-Content Analysis Software.

Undifferentiated hPSCs were seeded onto Matrigel-coated dishes at a density of 4 × 10⁴ cells/cm² and allowed to expand for 48 h (∼80% confluency). At this stage (d1 of differentiation), cultures were treated with medium comprising StemPro34 supplemented with (1:100 dilution) Matrigel and (1 ng/mL) BMP4 (R&D systems) and after 24 h (d2 of differentiation), medium comprising StemPro34 with (10 ng/mL) BMP4 and (8 ng/mL) Activin A (Life Technologies). Medium exchange was performed on d4 of differentiation using RPMI supplemented with 1xB27 (Life Technologies) and small molecule inhibitors, KY02111 (10 μM) and XAV939 (10 μM) (R&D systems). From d8 onward, cells were maintained in RPMI medium supplemented with B27 only, with medium changes every 3 days. Cardiac differentiation efficiency was accessed by using immunocytochemistry with primary mouse anti-human α-actinin antibody (No. A7811, 1:800; Sigma) dilution and secondary goat anti-rabbit Alexa633 (No. A21052, 1:400; Invitrogen), counterstaining with 0.5 μg/mL DAPI (No. D9542, 1:500; Sigma). Immunofluorescence images were captured using Operetta High-Content System (Perkin Elmer) and analyzed using Harmony High-Content Analysis Software.