Ab155902

In total, 5 × 10³ cells were seeded in 96-well plates. Five to seven technical replicates per condition were used. After 24 h of culture, media was replaced with fresh media containing 10% WST-1 (ab155902; Abcam) and cells were left in the incubator for 1 h. Absorbance was read at 450 nm using the plate reader. For experiments in Supplementary Fig. 9, we used a very similar Cell Cytotoxicity Assay Kit (ab112118; Abcam). Here, after 24 h of culture, media was replaced with fresh media containing 20% ab112118 and cells were left in the incubator for 3 h. Absorbance was read at 570 and 605 nm using the plate reader, and their ratio was used to evaluate viablility/proliferation.

After transfection, cell proliferation was assessed using the WST assay. A total of 3 × 10³ transfected cells and negative control cells were seeded in 96-well plates from 1 to 7 days. On each of the mentioned days, cell proliferation was measured using WST-reagent (ab155902, Abcam). Seven percent of the WST reagent was added to each well with phenol red-free media. The plate was incubated for 4 h at 37°C. Then, absorbance was measured at 450 nm in a microplate reader with background correction at 650 nm. The significance of any differences were assessed using t-test.

The proliferation of wild-type (WT), shCtrl, and shTXNIP ARPE-19 cells was determined using a Wst-1 assay. Cells (1 × 10⁴ cells/well) were seeded into 96-well plates. After overnight incubation, the culture medium was removed, and the cells were rinsed with phosphate-buffered saline (PBS). The cells were treated with or without H₂O₂ in culture medium containing 5% FBS. After a certain period of time (24, 48, or 72 h), 10 μl of Wst-1 reagent (ab155902, Abcam, USA) was added to each well. After incubation with Wst-1 reagent for 2 h, the medium was collected, and the absorbance of the treated and untreated samples was measured using an ELISA microplate reader at 440 nm. Each experiment was performed in triplicate and repeated three times to assess the reproducibility of the results.

Fibroblasts (CRL-1490; ATCC) were expanded in Eagle’s Minimum Essential Medium (EMEM; ATCC) supplemented with fetal bovine serum (10%) at 37 °C in humidified 5% CO₂. Cells were seeded onto plates at 2 × 10⁵ cells/mL prior to exposure to coal particles at concentrations of 0, 50 or 200 µg/mL for 24 h. Cell proliferation was assessed by WST-1 (ab155902; abcam) assay according to the manufacturer’s instructions. The production of collagen by the cells in response to coal particle exposure was assessed by soluble collagen assay (Sircol) according to the manufacturer’s instructions. All experiments were repeated 6 times (fresh cell and coal preparations on different days) to allow statistical comparisons to be made between samples.

Cells (1 × 10³) were seeded in five, 96-well plates. Six technical replicates were used per condition. At consecutive time points, separated 24 h apart, complete media were replaced with fresh serum-free media containing 10% WST-1 viability and proliferation reagent (ab155902; Abcam), and cells were incubated for an additional 45–60 min. Absorbance was read at 450 nm using the plate reader.

The assays were performed in accordance with the manufacturer’s instructions. Briefly, a total of approximately 0.25 × 10⁵ cells were cultured in each well of a 96 well plate in a final volume of 100 µl. Triplicates of each condition were prepared. For MTT assay, After CO exposure, MTT (Thiazolyl blue tetrazolium bromide, MTT; Cat #: M5655, Sigma-Aldrich Canada, Oakville, Ontario) or WST-1 (#ab 155902, Abcam, Cambridge, UK) was added to each well, and the plates were incubated in a 5% CO₂ atmosphere at 37°C for 4 h. Then, the solution was removed, followed by the addition of 200 μl of DMSO to each well. The absorbance was detected at 570 nm for MTT assay or 460 nm for WST-1 assay on a plate reader. The experiments were performed in triplicate in three individual experiments.

Primary corneal epithelial cells were established following our publications (7 (link)). In brief, C57BL/6J corneas were excised under a dissection microscope and placed in a petri dish with dissecting medium on ice. After trimming off any iris, corneas were cut into four pieces and transferred as explants to a 48-well culture plate. After letting the cornea pieces adhere to the bottom of the well, SHEM medium was added (DMEM + F12 + 10%FBS with EGF 5 ng/ml, cholera toxin A subunit 30 ng/ml, hydrocortisone 21-hemisuccinate sodium salt 0.5 ug/ml, 0.5% DMSO, 1X ITS, gentamicin 50 ug/ml, and amphotericin). Explants were incubated at 37°C with 5% CO₂. When cornea epithelial cells had grown to almost confluent, pan and C1 were added at 10, 50, and 100 μM, respectively. WST-1 reagent was added at 1:10 according to the company protocol (Abcam, Cat# ab155902). The absorbance of untreated (control) and treated wells was monitored in a microplate reader (OD = 420–480 nm, 0, 2, 4, 6, and 24 h, reference wavelength 650 nm).