4.4. Lactate Dehydrogenase (LDH) Cytotoxicity Assay

The measurement of the amount of LDH released from cells is one of the major methods to assess cell death, especially necrotic cell death. Released lactate dehydrogenase (LDH) activity was measured using an LDH cytotoxicity assay kit (DoGenBio Co., Seoul, Korea). Briefly, 1 × 10⁵ cells in a six-well plate (BD Falcon) were incubated with ATO and VPA alone and in combination with ATO/VPA for 24 and 72 h. After treatment, culture media was collected and centrifuged for 5 min at 1500 rpm. The supernatant was dispensed into a 96-well plate, and LDH assay reagent was added to each well. The plate was incubated at room temperature for 30 min. Absorbance values were measured at 450 nm using a microplate reader (Synergy^TM2). LDH release was expressed as the percentage of extracellular LDH activity compared with the control group.