Cell proliferation kit

The effect of Pyrrothiogatain on Calu-3 cell proliferation was determined using the XTT assay (Cell Proliferation Kit, 20-300-1000, Biological Industries, Bet Haemek, Israel) as per the manufacturer’s instructions. Briefly, 10⁵ Calu-3 WT cells were seeded in 96-well plate. Twenty-four hours later, the cells were incubated with Pyrrothiogatain at different concentrations (150, 300, and 500 µM) for an additional 48 h. Then, XTT reagent was added for 15 min before reading the change in absorbance at 650 and 475 nm using the SpectraMax 250 plate reader (Molecular devices). Specific absorbance measurements were given as the mean ± SD absorbance calculated from two repeat wells/samples. The mean specific absorbance was normalized at each time point to that of the non-treated control.
For crystal violet staining, following 48 h of SARS-CoV-2 infection the medium was removed from the cells that were then fixed and stained with 0.1% crystal violet solution (Biological Industries, Israel) for 5 min. Then stain was aspirated and plates were rinsed once with tap water and air-dried.

Cell proliferation activity was determined by XTT assays using a Cell Proliferation Kit (Biological Industries, Beit-Haemek, Israel). Cell migration ability was evaluated with wound healing assays. Cell invasion ability was determined with Corning Matrigel Invasion Chambers (Discovery Labware, Inc., Bedford, MA, USA). Detailed procedures were described in our earlier reports [16 (link),20 (link)].

SARS-CoV-2 (GISAID accession EPI_ISL_406862) was kindly provided by Bundeswehr Institute of Microbiology, Munich, Germany. Virus stocks were propagated (four passages) and titered on Vero E6 cells (Vero E6, ATCC^® CRL-1586™). Handling and working with SARS-CoV-2 virus were conducted in a BSL3 facility in accordance with the biosafety guidelines of the Israel Institute for Biological Research (IIBR). Vero E6 cells were seeded at a density of 3 × 10⁴ cells per well in 96-well plates. After overnight incubation, cells were treated in three replicates with Torin-1 (Fisher Scientific) or Remdesivir (Medchemexpress) as indicated. Cells were infected 1 h later with SARS-CoV-2 (MOI, 0.01–0.015). Cell viability was determined 72 h after infection by using the Cell Proliferation Kit (XTT based, Biological Industries, Israel) according to manufacturer's protocol. For positive control, cells were treated with Remdesivir, for negative control cells were not treated prior to infection.

To examine cell viability of astrocytes and BECs subjected to the OGD treatment, 5 × 10³ cells were seeded overnight in coated 96 well-tissue plates. Cells were washed once with PBSX1 and exposed to OGD treatment or normoxia for 4 h with 100 μl relevant DMEM starvation medium. In these studies, AM is astrocyte medium (cat. no. 1801 ScienCell Research Laboratories) and EBM2 is Endothelial Cell Growth Basal Medium-2 (cat. no. 00190860 Lonza). After 4 h the medium was replaced with EBM-2 or AM containing 0.5% serum. Cell viability was determined after 44 h by using Cell Proliferation Kit (XTT, Biological industries, Kibbutz Beit Haemek, Israel), according to the manufacturer’s instructions. Absorbance at 450 nm (OD₄₅₀) was determined for each well using an automated microplate reader (SpectraMax 190; Molecular Devices, Sunnyvale, CA, United States) and subtraction of non-specific readings (measured at 630 nm) was done automatically during the OD₄₅₀ reading.

First, 6 × 10³ microglia cells were seeded on a collagen-coated 96-well plate for 24–72 h. Alternatively, 5 × 10³ MBM cells were seeded on a 96-well plate for 24 h, then treated with microglia CM for 48 h. Cell viability was determined using a Cell Proliferation Kit (XTT, Biological Industries). Cell viability at 24 h after seeding was determined as time point 0. To obtain the relative cell viability, the optical density (OD) of each cell variant (at each time point) was divided by the OD of the corresponding variant at time point 0.

For cytotoxicity assays, 100 μL of cell suspension (approximately 2 × 10³ cells·mL⁻¹) was seeded in each well of a 96‐well plate, and cells were allowed to attach overnight. The next day, culture media were discarded, and the cells were treated with different concentrations of TAK‐931 (catalog no.: HY‐100888/CS‐0020550; MedChemExpress, NJ, USA) ranging from 1 to 1000 nm. The plates were incubated for 5 days, and media containing TAK‐931 were replaced every other day. Cells treated with 0.1% DMSO served as a negative control. To investigate cell toxicity, XTT assays were performed using a Cell Proliferation Kit (Biological Industries) according to the manufacturer's protocol. The signals were measured using a microplate reader (Bio‐Rad Laboratories, Inc.).

Cell Proliferation Kit (XTT, Biological industries) was used according to the manufactures’ instructions. To obtain the relative cell viability, the optical density (OD) of the treated cells (in each time point) was divided by the OD of the non-treated cells at the same time point of the experiment.