LDHReleaseTests

LDH release tests, also referred to as LDH cytotoxicity assays, are in vitro methods used to evaluate cell membrane integrity and cytotoxicity. They measure lactate dehydrogenase activity outside cells when the enzyme leaks into culture medium following cell damage.

Principle: LDH is a stable cytosolic enzyme released upon plasma membrane damage. In the assay, the sample medium is mixed with a reaction system containing lactate and NAD+. LDH converts lactate to pyruvate, reducing NAD+ to NADH. NADH then participates in a secondary reaction that reduces a tetrazolium salt to a colored formazan or generates a fluorescent signal, allowing quantification of LDH activity.

Typical protocol: treat cells, collect supernatant at chosen time points, and include low (spontaneous) and high (max) release controls. Add the assay reagents, incubate for a defined period, and measure absorbance (often ~490 nm) or fluorescence. A separate lysis step provides the maximum release control.

Data interpretation: cytotoxicity is expressed as a percentage of total LDH release, commonly calculated as (experimental - low)/(high - low) × 100. Results depend on cell type, serum conditions, plate type, incubation time, and handling; background LDH and spontaneous release must be considered.

Applications and limitations: LDH release assays are simple, non-radioactive, and suitable for high-throughput screening of cytotoxic compounds. Limitations include lack of specificity for necrosis vs late apoptosis, potential interference from serum components, LDH stability, and the need for proper controls to distinguish true cytotoxicity from basal LDH release.