Lactate dehydrogenase cytotoxicity assay kit

Cellular viability was assessed using an MTT assay and was expressed as a percentage of the absorbance value at 562 nm of the control cells. Cell death was assessed by using a lactate dehydrogenase cytotoxicity assay kit according to the manufacturer’s instructions (Beyotime Biotech, Nanjing, China), and the absorbance value of each sample was read at 490 nm. The cell death percentage was calculated by using the following formula: cell death percentage (%) = (Asample − Acontrol)/(Amax − Acontrol) × 100, where Asample is the sample absorbance value; Acontrol is the absorbance value of the control group; and max is the absorbance value of the positive group.

Cells were inoculated into 24‐well plates at 5 × 10⁵ cells well⁻¹ overnight and dosed for 48 h. The plates were centrifuged at 400 g for 5 min using a multi‐well plate centrifuge. 120 µL of supernatant from each well was carefully aspirated into a 96‐well plate and subsequently assayed according to the instructions of the Lactate Dehydrogenase Cytotoxicity Assay Kit (Beyotime, China). The measured data were analyzed and processed with GraphPad Prism 8.0. Each experiment was repeated three times.

Cell mortality was determined using a lactate dehydrogenase cytotoxicity assay kit (Beyotime, Shanghai, China). According to the instructions, the absorbance value of each well was determined at 490 nm, and the mortality rate was calculated according to the formula: cytotoxicity or mortality (%) = (treatment sample absorbance − sample control well absorbance) / (absorbance of the maximum enzyme activity of cells—sample control well absorbance) × 100%.

Human corneal epithelial cells (HCEC) were purchased from BeNa culture collection (Jiangsu, China), maintained in Minimum Essential Medium (MEM; XP Biomed Ltd., Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, New York, NY, USA) and cultured in 60 ml flasks kept at 37°C in a humidified incubator containing 5% CO₂.
For in vitro cytotoxicity assay, the cultured HCEC were co-cultured with F. oxysporum conidia for 24 h (Kolar et al., 2017 (link)) in 96-well plates (1 × 10⁴ cells/well), then the lactate dehydrogenase (LDH) released from the cultured HCEC was measured using a lactate dehydrogenase cytotoxicity assay kit (Beyotime, Jiangsu, China; Jin et al., 2007 (link)).
An in vivo virulence assay was performed with AB strain zebrafish (ZFIN ID: ZDB-GENO-960809–7) as previously described (Laanto et al., 2012 (link)). Briefly, zebrafish (three-day post-fertilization) were infected with the F. oxysporum conidia by bathing. The fish were individually challenged with 1 × 10⁴ CFU/ml conidia, and survival was recorded every 12 h. All the experiments in this study were approved by the animal ethics committee of Jilin University.

U87 (5 × 10⁴ cells/well), U251 (5 × 10⁴ cells/well), SHG-44 (5 × 10⁴ cells/well), and C6 (1 × 10⁵ cells/well) glioma cells were treated with target compounds after being seeded onto 96-well microplate with six duplicated wells and cultured 24 h. Cellular viability was assessed using an MTT assay, and was expressed as a ratio to the absorbance value at 570 nm of the control cells. Cell death was assessed by using lactate dehydrogenase cytotoxicity assay kit according to the manufacturer’s instructions (Beyotime Biotech, Nanjing, China), the absorbance value of each sample was read at 490 nm, and cell death ratio was calculated by using the following formula: cell death ratio % = (A sample − A control/A max − A control) × 100. A sample: sample absorbance value; A control: the absorbance value of control group; A max: the absorbance value of positive group.

LDH release was detected with a lactate dehydrogenase cytotoxicity assay kit (C0017, Beyotime, China). Briefly, HPASMCs were cultured in 96-well plates until the cells proliferated to approximately 60% confluence and then were treated with DSF (10 μmol/L) for 24 h with or without hypoxia. The cell culture supernatant (120 μL) was collected and mixed with 60 μL of substrate and then incubated for 30 min at r.t. The OD490 was measured with a full-wavelength microplate reader (Thermo Fisher).