Lactate dehydrogenase assay kit

Cells were harvested, and the activity of HK and LDH was performed by Hexokinase Activity Assay Kit (Abcam, USA) and Lactate Dehydrogenase Assay Kit (Abcam, USA) according to the manufacturer's protocols. In brief, the cells were collected by trypsin and washed 3 times with precooled PBS, resuspended in precooled assay buffer, centrifuged at 12,000 rpm at 4 °C for 5 min to remove insoluble materials. The supernatant was collected, mixed with the reaction mixture, and incubated at room temperature in the dark for 40 min. The absorbance at 450 nm was measured by the microplate reader (BD, USA) and calculated the relative activity.

Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme present in most eukaryotic cells, and is released upon cell death due to damage to the plasma membrane. LDH activity was measured in samples of plasma using the Lactate Dehydrogenase Assay Kit (Abcam; Cambridge, UK). In this assay, LDH reduces NAD to NADH which then interacts with a specific probe to produce color. Changes in absorbance due to NADH formation were recorded at 450 nm at 37 °C using an automated microplate reader (iEMS Reader MF; Labsystems, Helsinki, Finland). The activity of the samples was expressed in milliunits per milliliter (mU/mL). All samples were run in duplicate. The lower limit of detection for ELISA ranged from 14 to 36 mU/mL.

According to the manufacturer's protocols, a Lactate Dehydrogenase Assay Kit (Abcam, Cambridge, UK) was used to measure the release of LDH in supernatants following stimulation of cells.

Caspase-3 activity was measured by using the EnzChek™ Caspase-3 Assay kit purchased from Invitrogen Molecular Probe, using the manufacturer's instructions. Briefly, about 6 × 10⁵ cells were washed with PBS and then lysed in 65 μl of the kit lysis buffer. The caspase-3 substrate Z-DEVD-AMC was added to a final concentration of 200 μM in 50 μl of the kit reaction buffer, which was then mixed to an equivalent volume of cell lysate. Fluoromethylcoumarin fluorescence, resulting from the caspase-3-mediated cleavage of Z-DEVD-AMC, was detected using the POLARstar Omega microplate reader (BMG Labtech) with excitation/emission wavelengths of 350/450 nm with a 45-min kinetics. The remaining cell lysate was used to normalize the fluorescence values to the total protein content. Results were expressed in comparison to nonsteatotic or steatotic untreated cells, as appropriate. Lactate dehydrogenase (LDH) release in the culture medium was used as an indicator of cell necrosis, as previously reported [23 (link), 24 (link)]. To this end, LDH activity was measured in culture medium aliquots using the Lactate Dehydrogenase Assay Kit purchased from Abcam, according to the manufacturer's recommendations. Results were expressed in comparison to nonsteatotic untreated cells.