Varioskan lux plate reader

HEK293T cells were seeded in 96 well plates and transfected with either a plasmid encoding the full-length SARS-CoV-2 spike (pLVX-EF1alpha-SARS-CoV-2-S-2xStrep-IRES-Puro) or mock plasmid (pcDNA3.1) using branched PEI (Sigma). Media was switched 24 h post-transfection. At 48 h post-transfection, cells were washed 5 times with PBS prior to fixation with 4% paraformaldehyde in media for 30 min at 4 °C. All further washing steps were performed 5 times with PBS + 0.05% Tween 20 (PBS-T). Cells were washed prior to blocking in blocking buffer (PBS-T + 2% BSA) for 1 h at room temperature. After washing, cells were incubated with dilutions of primary antibodies in blocking buffer for 2 h at room temperature. After washing, cells were incubated with goat anti-human IgG (Jackson ImmunoResearch) at a 1:5,000 dilution in blocking buffer for 1 h at room temperature. After washing, substrate solution (Pierce 1-Step) was used for colour development according to the manufacturer's specifications. Optical density at 450 nm was read on a Varioskan Lux plate reader (Thermo Fisher Scientific). The difference in signals between cells transfected with full-length spike and mock plasmid was calculated.

Olivacine, similar to ellipticine, is a compound with fluorescent properties. Therefore, by analyzing the intensity of fluorescence over time, it is possible to assess the cellular pharmacokinetics of the compounds. At the same time, cell morphology was also assessed.
The cells were seeded into a 96-well plate and cultured for 24 h (37 °C, 5% CO₂) for cell adhesion to the surface and regeneration. The following day, olivacine or ellipticine was added for 1 h at a concentration of 10 µM. The culture plate was placed under a Juli microscope, and the device with the plate inside was put in the CO₂ incubator. Pictures were taken every 15 min for 24 h. We have prepared software that allowed us to measure the fluorescence intensity of the indicated cells. Fluorescence analysis was performed for all images (every 15 min) during a period of rapid change in fluorescence intensity and less often at a later period when it stabilized. Three independent experiments were carried out, always analyzing 50 selected cells.
When the cells were incubated with the test compound, the supernatant took 2 μM every 15 min into another microplate, and fluorescence was measured using a Varioskan LUX plate reader (Thermo Fisher Scientific) to assess whether the concentration of test compounds increased in the medium.

To quantify cell proliferation in low serum medium and the four treatment groups, cells were plated in a 24-well plate 7,000 cells/cm². They were left to grow for two days in each treatment and trypsinized using 0.25% trypsin-EDTA. The cell solutions were centrifuged at 300 xg for 5 minutes and resuspended in 1x PBS with Hoechst dye (Life Technologies 33342) at a concentration of 5 mg/mL. The cell solutions were transferred to a 96-well plate and fluorescence was measured at 390 nm on a Thermo Scientific Varioskan LUX plate reader.

RBC glutathione levels were measured using a GSSG/GSH Quantification Kit (Dojindo) according to the manufacturer’s protocol^{38 (link)}. Briefly, RBCs were hemolyzed with 10 times the amount of 5% 5-sulfosalicylic acid solution (FUJIFILM Wako), and the samples were centrifuged at 8000 × g for 10 min to remove proteins^{7 (link)}. The samples and buffer solution were mixed and incubated for 1 h at 37 °C. Then Substrate and Enzyme/coenzyme working solution was added. After 10 min of incubation, absorbance was measured at 412 nm using a Varioskan LUX plate reader (Thermo Fisher Scientific, Kanagawa, Japan). The number of mice in each group is shown in Supplementary Table S1.