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In This Article

  • Overview
  • Protocol
  • Results
  • Disclosures
  • Materials

Overview

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-results-58
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, respectively. Both t.......

Disclosures

No conflicts of interest declared.

Materials

NameCompanyCatalog NumberComments
EnzyChrom Pyruvate Assay KitBioAssay Systems#EPYR-100colorimetric/ fluorometric 100 assays; Store at -20°C
Trichloroacetic AcidWako Pure Chemical#207-04955store at room temperature
Teflon homogenizer Iwaki/Pyrex#358034 (Wheaton)Instead of Iwaki/Pyrex, available by Wheaton

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