Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms

Abstract

Source: Yanase, S. et al. Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans. J. Vis. Exp. (2018).

This video describes a colorimetric assay for quantitatively measuring pyruvate in the cellular fractions of Caenorhabditis elegans. Utilizing the hydrogen peroxide produced from the enzymatic reaction of pyruvate oxidase with cellular pyruvate, a chromogenic dye is oxidized to produce a colored product, measured using colorimetry to determine the pyruvate concentration.

Protocol

1. Synchronized Culture of C. elegans

1. 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.
   1. 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).
2. To make nematode growth medium (NGM) agar, use 3 g....

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)

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EnzyChrom Pyruvate Assay Kit	BioAssay Systems	#EPYR-100	colorimetric/ fluorometric 100 assays; Store at -20°C
Trichloroacetic Acid	Wako Pure Chemical	#207-04955	store at room temperature
Teflon homogenizer 	Iwaki/Pyrex	#358034 (Wheaton)	Instead of Iwaki/Pyrex, available by Wheaton