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Colorimetric Assay for Quantification of Lactate in Test Samples
Overview
This video describes a colorimetric assay for lactate measurement in cell lysates by using the lactate dehydrogenase enzyme. This enzyme converts lactate into pyruvate, along with the conversion of NAD to NADH that reacts with tetrazolium dyes and gives a colored product that can be measured spectrophotometrically to quantitate the lactate.
Protocol
1. Synchronized Culture of C. elegans
- Before seeding, culture the Escherichia coli (E. coli) strain OP50 overnight at 37 °C in 300 mL of Luria-Bertani (LB) broth liquid medium. Store the cultured OP50 at -4 °C.
- To make LB broth liquid medium, use 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1.5 mL of 1 N NaOH, and add to 1 L with deionized water. Autoclave.
NOTE: OP50 and C. elegans strains are available from the Caenorhabditis Genetics Center (University of Minnesota, St. Paul, MN, USA).
- To make LB broth liquid medium, use 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1.5 mL of 1 N NaOH, and add to 1 L with deionized water. Autoclave.
- To make nematode growth medium (NGM) agar, use 3 g....
Representative Results
Figure 1. Process for the extraction of cellular metabolites. (A) 1,500-3,000 worms were placed on an NGM agar plate (90-mm Petri dish). Extraction from worms on five plates was sufficient for the colorimetric assays. (B) Worms collected from the five plates of 3,000 and 1,500 worms per plate are indicated on the left and right side of panel, res.......
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Source: Yanase, S., et al. Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans. J. Vis. Exp. (2018).
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