Operetta high content system

The infected macrophages were fixed with a paraformaldehyde 4% in phosphate buffered saline during 20 min at room temperature, after 72 h of treatment. Cells were stained with Hoechst 33342 (Thermo Fischer Scientific, Waltham, MA, USA) at 16 µM. Images were acquired using the Operetta High-Content System in non-confocal mode and 20X air objective, for further segmentation and quantification (Perkin Elmer, Waltham, MA, USA). Pictures from five fields per well were analysed for reliable statistics.

HEK293 cells (4 × 10⁴ cells/well), N2a cells (4 × 10⁴ cells/well), or mPN cells (3 × 10⁴ cells/well) were seeded onto 96-well plates. Cells were pretreated with FAC (Macklin, A800010) at the indicated concentrations for 1 h at 37°C. The cells were next infected with ERA-EGFP (MOI = 0.05) for 1 h at 37°C, thoroughly washed, and then cultured with 2% FBS-containing medium. FAC was present in the culture medium throughout the infection. At 48 h postinfection, the cells were fixed with 3% paraformaldehyde, and the cell nuclei were stained with Hoechst 33342. The infection ratio was then determined by using the Perkin-Elmer Operetta high-content system.

HEK293 cells (4 × 10⁴ cells/well), N2a cells (4 × 10⁴ cells/well), or mPN cells (3 × 10⁴ cells/well) were seeded onto 96-well cell carrier plates (Perkin-Elmer, 6055302). The media were removed from the well, and the cells were treated with the indicated concentrations of TfR1 antibody (BD Pharmingen, 555534), or isotype antibody IgG2a (20 μg/mL; Southern Biotech, 0103-01) for 1 h on ice. Cells were then incubated with ERA-EGFP (MOI = 0.05) for 1 h at 4°C in the presence of the indicated concentrations of antibody. They were then washed and incubated with medium containing the indicated antibodies at 37°C. At 48 h postinfection, the cells were fixed with 3% paraformaldehyde, and the nuclei were stained with Hoechst 33342. The infection ratio was determined by using the Perkin-Elmer Operetta high-content system.

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.

HEK293 cells (4 × 10⁴ cells/well), N2a cells (4 × 10⁴ cells/well), or mPN cells (3 × 10⁴ cells/well) were seeded onto 96-well carrier plates. ERA-EGFP (MOI = 0.05) was mixed with the purified soluble TfR1-GST protein at the indicated concentrations or with GST at 4°C for 1 h. The media were removed from the well, and the cells were incubated with the virus-protein mixture at 37°C for 1 h. The cells were then washed and incubated with growth medium. At 48 h postinfection, the cells were fixed with 3% paraformaldehyde, and the nuclei were stained with Hoechst 33342. The infection ratio was determined by using the Perkin-Elmer Operetta high-content system.