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

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 of NaCl, 2.5 g of peptone, 17 g of agar, and 975 mL of deionized water. Autoclave. Cool to 55 °C, then sterilely add, in order, 1 mL of 1 M MgSO₄, 1 mL of 1 M CaCl₂, 1 mL of 5 mg/mL cholesterol in EtOH, and 25 mL of 1 M potassium phosphate pH 6.0 in 90-mm Petri dishes.
   1. For 1 M potassium phosphate pH 6.0, use 108.3 g of KH₂PO₄ and 46.6 g of K₂HPO₄, and add deionized water to 1 L. Autoclave.
3. Spread 1-2 mL of the cultured OP50 on the NGM agar plates. To make a thin layer of OP50, incubate the plates overnight at room temperature before adding any nematodes.
   NOTE: The NGM agar plates inoculated OP50 can be stored at room temperature for 2-3 weeks.
4. Add at least 100 worms onto an NGM agar plate with OP50, and culture at 20 °C until the adult stage. At least three plates are required.
5. To collect eggs in utero, transfer gravid hermaphrodites from the three NGM agar plates each with 5 mL of S buffer in a 15-mL conical tube, and wash the worms 3 times with 15 mL of S buffer using centrifugation at 300 x g for 30 s at room temperature.
   1. To make S buffer, use 5.9 g of NaCl and 50 mL of 1 M potassium phosphate pH 6.0, and add to 1 L with deionized water. Autoclave.
6. Dissolve the worms in an alkaline hypochlorite solution for axenization and bulk egg isolation (0.5 mL of fresh bleach or equivalent: 5-6% sodium hypochlorite, 0.1 mL of 10 M NaOH, approximately 4.5 mL of S buffer), and stand the solution for 10-15 min at room temperature with mixing by inverting.
   NOTE: Within 15 min, adult worms should dissolve, leaving a hazy solution of eggs liberated from their carcasses (the liberated eggs should be confirmed using a stereoscopic microscope). Instead of 0.1 mL of 10 N NaOH, 0.2 mL of 5 N NaOH can also be used.
7. After hypochlorite treatment, wash the egg pellet 3 times with 15 mL of S buffer, and resuspend in 5-6 mL of S buffer. Hatch the released eggs during an overnight incubation at 20 °C in S buffer without E. coli for an age-synchronous culture of L1 stage larvae.
8. To determine the approximate number of L1 stage larvae, count the worms using a stereoscopic microscope in 10 µL of S buffer after resuspending the larvae at least 3 times, and calculate the average. Then, transfer the L1 stage larvae to five NGM agar plates with OP50 (1,500-3,000 worms per plate using a 90-mm Petri dish), and culture at 20 °C until they have grown to the young adult stage when the self-fertilization begins and a few eggs are laid (ordinarily after 3 days).

2. Extraction of Cellular Fraction from C. elegans

1. Collect the young adult stage (5-day-old animals) worms from the five NGM agar plates with S buffer (Figure 1A).
   1. To select only living worms using the sucrose method 6 for flotation on 30% (w/v) sucrose, mix the worms suspended in 3-4 mL of S buffer with an equal volume of ice-cold 60% (w/v) sucrose in a 15-mL conical tube. Spin the tube at 1,500 x g for 15 s at 4 °C and remove the floating worms into a fresh tube by moving them off the wall of the tube with a Pasteur pipette.
2. Wash the worms 3 times with S buffer by centrifugation at 1,500 x g for 30 s at 4°C. Check the wet volume of washed worms after centrifugation using a 1,000 µL micropipette tip (Figure 1B).
3. Add the washed worms to an equal volume of ice-cold 10% (w/v) trichloroacetic acid (TCA; final concentration of 5%) for protein precipitation (Figure 1C). Instead of TCA, perchloric acid (PCA) or metaphosphoric acid can be used.
4. Homogenize the worms with the precipitant using 40 strokes of a pestle in a Teflon homogenizer (Potter-Elvehjem tissue grinder) with rotation at up to 1,300 rpm on ice.
5. Transfer the homogenate into a fresh 1.5-mL microtube with a Pasteur pipette, and sonicate using an ultrasonic homogenizer for 3 min (3 times of 1 min) with a 20% duty cycle on ice.
6. Clarify the homogenate by centrifugation at 8,000 x g for 10 min at 4 °C. Neutralize the supernatants with 4 M KOH (0.25 volume to 10% TCA) for 20 min on ice, and centrifuge at 8,000 x g for 10 min at 4 °C. The supernatant (as a test sample) can be stored at -80 °C until the following assays.

3. Pyruvate Assay Using a Colorimetric Assay Kit

1. Measure the concentration of pyruvate in the supernatants (test samples) using a colorimetric assay kit (Table of Materials). Carry out duplex examinations for the test samples. Add 10 µL of the test samples and 90 µL of Working Reagent (containing 94:1 of Enzyme Mix and Dye Reagent, which are provided with the kit) into a 96-well plate and incubate at room temperature for 30 min while the samples are protected from light.
2. Measure the absorbance of each well at 570 nm using a microplate reader.
3. Plot the pyruvate standard curve. Calculate the pyruvate concentrations of the test samples from the pyruvate standard curve.

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 the wet volumes in each 15-mL tube are < 0.5 mL, which were sufficient for detection. (C) Protein precipitation using 10% TCA during homogenization of the worms. Adding the worms into ice-cold 10% TCA in a homogenizer has to be performed before the worms are homogenized. Otherwise, cellular lactate and pyruvate cannot be detected in test samples using a colorimetric assay kit.