Ldh test

Release of lactate dehydrogenase (LDH) from the cytosol to culture medium is a marker of cell death. The assay was performed after 24 h incubation of HaCaT cells in 24-well plates with investigated concentrations of each compound. The activity of LDH released from the cytosol of damaged cells to the supernatant was measured using the protocol of the cytotoxicity detection kit LDH test described by the manufacturer (Roche Diagnostics, Berlin, Germany). Absorbance was measured at 490 nm using a microplate reader (using Epoch microplate reader, BioTek Inc., Winooski, VT, USA) equipped with Gen5 software (BioTech Instruments, Inc., Biokom, Janki, Polska)
Compounds mediated cytotoxicity expressed as the LDH release (%) was determined by the following equation: [(A test sample—A low control)/(A high control—A low control)] × 100% (A-absorbance); where “low control” were cells in DMEM with 2% FBS without tested compounds, and “high control” were cells incubated in DMEM with 2% FBS with 10% Triton X-100 (100% LDH release).

The LDH test (Roche) was performed with medium samples from OMICS-ALI according to the manufacturer’s instructions. Cell culture medium kept at the same CO₂ levels as exposed cells was used as a blank. Non-exposed control cells were lysed with 1% Triton X-100 for 20 min prior to the end of exposures, and the relative LDH content was used as the highest LDH release achievable; the absorbance values were set as 100% toxicity. Measurements were performed using a VICTOR3™ multilabel plate reader (model 1420–051, PerkinElmer, USA).