Lactate dehydrogenase ldh assay

Cells (1 × 10⁵) were plated in the 24-well plate with 500 μL of medium, and were allowed to grow to 80%–90% confluence. HepG2 cells were exposed to different concentrations of K₂Cr₂O₇ for 24 h. Cr(VI) working solution was prepared in Dulbecco’s Modified Eafle’s medium (DMEM) to reach final concentrations of 0, 5, 10, 20, 40, or 80 μM. Resazurin reduction assay was performed to determine cell viability after Cr(VI) treatment. Fifty microliters of resazurin (1 mg/mL stock) was added to the 24-well plates (500 μL/well), which were incubated for 2 h at 37 °C. Fifty microliters of color changed medium was transferred from each well to a 96-well plate and fluorescence was measured at 530 nm/590 nm (excitation/emission), using a multi-well fluorometric reader CytoFluor series 4000 (Applied Biosystems, Drive Foster, CA, USA). Lactate dehydrogenase (LDH) assay (Promega, Madison, WI, USA) was performed to determine cytotoxicity after Cr(VI) treatment. Fifty microliters of medium was transferred from each well to a 96-well plate and 50 μL of substrate mix was added to each well. The 96-well plate was then incubated for 30 min at room temperature. Subsequently, 50 μL of stop solution was added to each well and absorbance was measured at 560 nm using a plate reader. Data are presented as mean ± SEM. Three independent experiments in three replicates were performed.

At passage 3–5, PAFs were grown to confluence in DMEM10% fetal calf serum (FCS) (Invitrogen, Burlington, ON, Canada) on collagen I (Corning, New York, NY, USA) coated 6-well plates. Cells were serum-deprived overnight and stimulated with/without 1 ng/mL recombinant IL-1α/β or IL-33 for 24 hours, which was determined following dose experiments (see Supplementary Fig. S14). Cell-culture supernatant, protein-lysates and RNA were harvested for ELISAs, western blots and qRT-PCR.
collagen I gels were made as previously described^{23 (link)}. 50,000 PAFs were seeded on top and allowed to migrate into and contract the gels. Cells were stimulated with 10 mg/ml β-aminopropionitrile (BAPN) (see Supplementary Fig. S15) or 1 ng/ml IL-1α,-β or IL-33 for 24 hours. Cell-seeded gels were assessed for contraction using Image J software and gel weight using a fine balance^{26 (link)}. A lactate dehydrogenase (LDH) assay (Promega, Madison, USA) to determine cell viability after cell stimulation, was done according to the manufacturer’s instructions.