C20300

The effect of different concentrations of CoCl₂ on the viability of HRECs was assessed by lactate dehydrogenase (LDH) Cytotoxicity Assay (CyQUANT™; Invitrogen-C20300, Waltham, MA, USA). In this assay, HRECs were cultured in 96-well plates (1 × 10⁴/well), and after cells became confluent, the culture media were replaced by media free of serum and growth factors for 10–12 h before applying different concentrations of CoCl₂ (0, 10, 100, and 1000 μM) for 24 h. After that, the amount of LDH released into the medium was determined per the manufacturer’s instructions.

HTM5 cells were plated in serum-free fresh medium in 96-well plates (Sarstedt) at a density of 40 000 cells/well (n = 6 wells/group) and treated with 20 ng/mL of PDGF, 5–15 µM FLU (PDGF + FLU), 5–15 µM SA-4503 (PDGF + SA; SA−4503: 165377-44-6 Tocris Bioscience, Bristol, UK) and 5–15 µM PRE-084 (PDGF + PRE; PRE−084: P2607 Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Control cells were treated with vehicle (HCl) alone. After the treatment period, cells were incubated with methyl-thiazoletetrazolium (MTT, M6494 Invitrogen, Carlsbad, CA, USA) for 4 h followed by solubilization in DMSO-ethanol (1:1). The formation of water-insoluble formazan was determined by measuring optical density at 570 nm in a Plate CHAMELEON™ V Fluorometer-Luminometer-Photom reader (Hidex, Turku, Finland). Lactate dehydrogenase (LDH) assay (C20300, Invitrogen, Carlsbad, CA, USA) was performed from the supernatant of the cells according to the manufacturer’s protocol, and samples were measured in the same way as in case of MTT.

HTM5 cells were plated in 96-well plates (40,000 cells/100 µL/well, n = 6/treatment group) and treated as mentioned above (Section 2.3). To investigate cell proliferation, after 24 h of various treatments, cells were incubated with methyl-thiazolyldiphenyl-tetrazolium bromide (MTT, 0.5 mg/mL, M6494, Invitrogen) for 3 h at 37 °C. Then, MTT was discarded, and 100 µL of solubilizer (dimethyl sulfoxide (DMSO, D4540, Sigma-Aldrich) was mixed in a 1:1 ratio with ethanol) was added to each well to dissolve the water-insoluble formazan crystals. The absorbance of the formazan solution was recorded at 570 nm (SpectroStar Nano microplate reader, BMG Labtech, Ortenberg, Germany). To assess cell toxicity, the widely used lactate dehydrogenase (LDH) assay (C20300, Invitrogen, Carlsbad, CA, USA) was performed from the supernatant of the cells according to the manufacturer’s protocol and the samples were measured in the same way as in the case of MTT.

CD4⁺ T cells were cultured with anti-CD3/CD28 beads. The LDH assay was performed according to the manufacturer’s instructions (C20300) from Invitrogen, (California, USA). Relative LDH release was calculated as LDH release [%] = 100 × (experimental sample LDH activity − spontaneous LDH release activity)/(maximum LDH release activity − spontaneous LDH release activity).

The LDH release quantification was performed using a colorimetric CyQUANT LDH Cytotoxicity Assay (Invitrogen, C20300, Carlsbad, CA, USA). Lactate dehydrogenase (LDH) is a cytosolic enzyme that is released into the cell culture medium upon the disruption of the plasma membrane, indicating the necrotic type of death. LDH is quantified in the media in enzymatic reactions. Firstly, LDH catalyzes the conversion of lactate to pyruvate with the accompanying reduction of NAD⁺ to NADH. Then, the added diaphorase oxidizes NADH, which leads to the reduction of a tetrazolium salt to a red formazan. The amount of formulated formazan is directly proportional to the total LDH released into the media. Here, cells (1 × 105/mL) in the log growth phase were seeded in a 24-well plate in the presence or absence of NAMPT inhibitors. At each time point, 100 µL of cells was transferred to a 96-well plate and the reaction mixture from the kit was added. The plate was then incubated at RT for 30 min and protected from light. Afterwards, the stop solution was added and the absorbance was measured at 490 nm with a spectrophotometer. The higher the absorbance intensity in the sample, the more LDH is released to the culture medium.