This article describes a method for quantifying intracellular lactate concentration in nematode cell lysates under anaerobic conditions. The process involves using lactate dehydrogenase to convert lactate to pyruvate, allowing for accurate measurement of lactate levels through a colorimetric assay.
In living cells, under anaerobic conditions, the enzyme lactate dehydrogenase converts pyruvate — an end product of glycolysis — to lactate — a key metabolite. The amount of lactate produced in the cells reflects their metabolic status.
To quantify the intracellular lactate concentration, begin with a test sample of protein-free, nematode cell lysate. Supplement other wells of a multi-well plate with a lactate solution of various known concentrations.
The lysate lacks all proteinaceous enzymes, including endogenous lactate dehydrogenase, ensuring accurate lactate quantification.
Add a lactate assay buffer into each well to maintain the appropriate pH for optimum enzymatic activity. Supplement the wells with a reaction mix containing the lactate dehydrogenase enzyme, nicotinamide-adenine dinucleotide — NAD — a coenzyme, and a colorimetric probe and incubate.
During incubation, lactate dehydrogenase catalyzes the oxidation of cellular lactate to pyruvate, with a simultaneous reduction of NAD into NADH. The generated NADH reacts with the colorimetric probe to produce a purple product.
Spectroscopically measure the purple solution's absorbance for the test sample and lactate standards at 570 nanometers. Plot the absorbance values of the lactate standards and generate the calibration curve.
Finally, compare the absorbance value of the sample with the lactate standards to determine the amount of lactate present in the test sample.
To measure the concentration of lactate, use a colorimetric assay kit to carry out duplicate analysis for the test samples. Add 10 microliters of the test samples to each well of a 96-well plate, and then add lactate assay buffer to each sample to adjust the volume to 50 microliters.
Dilute 100 millimolar L(+)-Lactate standard with lactate assay buffer to 1 millimolar. Then, into a series of wells, add 0, 2, 4, 6, 8, and 10 microliters of diluted L(+)-Lactate standard. Add 50 microliters of reaction mix into each well, and incubate at room temperature protected from light for at least 30 minutes for the color to develop.
Finally, use a microplate reader to measure the absorbance of each well at 570 nanometers. Subtract the absorbance of the background control mix from the absorbance of the reaction mix. Then, plot the lactate standard curve, and calculate the lactate concentrations from the curve by multiplying each concentration by 2.25.
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