Cytotoxicity Based Lactate Dehydrogenase Assay

Cytotoxicity or cell death is measured with assays using markers of apoptosis and/or necrosis of target cells (Saade et al., 2012; Specian et al., 2016). The colorimetric measurement of lactate dehydrogenase (LDH), is a reliable and commonly used technique (Specian et al., 2016). LDH, the cytosolic enzyme present in nucleated cells, is released into the extracellular media once the cell membrane is disrupted. The LDH activity is measured indirectly by the transformation of lactate in pyruvate with the reduction of nicotinamide adenine dinucleotide (NAD⁺) to NAD⁺H⁺ lactate (Specian et al., 2016; Abcam, 2019). The oxidation of the NADH⁺ that causes an increase in absorbance will be proportional to the LDH activity and therefore proportional to the number of lysed cells (Saade et al., 2012).

The LDH assay was performed according to the manufacturer’s suggested protocol (Abcam, 2019). H-Cytotox, 2019 (LDH Assay, Abcam® kit: ab65393). After addition of LDH reaction mix, an optimal 30 min incubation was used. The optical density of the plate was read with a FLUOstar Optima plate reader set at 450 nm with a 620 nm reference wavelength using the Optima version 2.1 software (BMG Labtech®). The percentage of cytotoxicity was calculated as per equation in Supplementary Figure S2. GraphPad Prism version 8.0 software was used for data analysis (GraphPad Software, La Jolla California United States, www.graphpad.com).