NADH-independent enzymatic assay to quantify extracellular and intracellular L-lactate levels

Lactate is a central metabolite in energy metabolism and is also involved in cell signaling and epigenetic regulations. Here, we describe an NADH-independent enzymatic assay allowing rapid, selective, and sensitive quantification of L-lactate down to the pmol range. We detail lactate extraction from intracellular and extracellular fractions, followed by total protein amount determination and enzymatic assay. This approach allows quantification of intracellular and extracellular L-lactate levels, validated by treating adherent and non-adherent cells with inhibitors of lactate transporters (MCT).