3.5.3. Lactate Dehydrogenase (LDH) Cytotoxicity Assay

Another test used to assess cytotoxicity of the tested hemp extracts was a high-throughput, reliable colorimetric method for quantifying cellular cytotoxicity. The activity of LDH in the studied extracts was determined using a commercially available kit (Cytoscan™ LDH Cytotoxicity Assay) from G-Biosciences (A Geno Technology, St. Louis, MO, USA). The assay is based on the conversion of lactate to pyruvate in the presence of LDH with parallel reduction of NAD. The test was carried out according to the instructions provided with the reagents. Analyzes were performed by seeding cells (keratinocytes and fibroblasts) into 96 well plates in DMEM medium. After attachment of the cells to the bottom of the wells, the plates were treated with MAE and UAE extracts at concentrations of 1–1000 μg/mL (Compound Treated). To prepare Spontaneous LDH Activity Control, sterile, ultrapure water was added to the wells instead of the tested extracts. To obtain Maximum LDH Activity Control, according to the instructions, 10 μL of Lysis Buffer was added to the wells. Following exposure the extracts diluted in DMEM, medium was removed and then the culture supernatant was collected and incubated with 50 μL reaction mixture. After incubation at room temperature for 30 min, the reaction was stopped by adding 50 μL Stop Solution. To determine LDH activity, absorbance at λ = 490 nm and λ = 680 nm was measured. Cytotoxicity of the analyzed extracts was calculated using the following equation: