Title:Identification of previously cryptic metabolic defects in the Complex II Caenorhabditis elegansmev-1 mutant strainusing metabolic flux analysis

Journal name: Biogerontology

Author list:Sheng Fonga,1, Li Fang Ngc, Li ThengNgb, Philip K. Mooreb, Barry Halliwella, and Jan Grubera,c*

aDepartment of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;(S. Fong);(B. Halliwell)

bDepartment of Pharmacology,Yong Loo Lin School of Medicine, National University of Singapore, Singapore; (L. T. Ng); (P. K. Moore)

c Yale-NUS College, Science Division, Singapore; (J. Gruber) ; (L. F. Ng)

*Corresponding author at: Yale-NUS College Research Laboratory, Science Division, Singapore; Block MD6, Centre for Translational Medicine (CeTM), NUS Yong Loo Lin School of Medicine, 14 Medical Drive, Level 10 South, 10-02M, Singapore 117599.

Tel.: +65 6601 3266. Email address: (J. Gruber).

1 Present address: Internal Medicine ResidencyProgramme, SingHealth Group, Singapore.

Supplementary Information

Metabolic flux analysis was performed in C. elegans using the XF96 Extracellular Flux Analyser (Seahorse Bioscience) according to the manufacturer’s instructions with the following modifications:

One day before the experiment:

  1. Add 200µl XF Calibrant into each well of a 96-well XF96 Utility Plate (part of a XF96 FluxPak).
  2. Hydrate the entire XF96 FluxPak overnight in the XF Prep Station at 37oC without CO2.

On the day of the experiment:

  1. Set up the XF96 Extracellular Flux Analyser.
  2. Given that the machine does not have temperature cooling control, leave the plate holder out and allow the machine to cool to room temperature (preferably between 20-25oC).

*Note the temperature configuration during run; ensure it does not rise significantly and does not reach temperatures harmful to the nematodes.*

  1. While leaving the XF96 Sensor Cartridge on top of the Utility Plate containing the calibrant,
  2. Add 25µl of 90µM FCCP (pre-diluted in M9 buffer from a 2.5mM stock dissolved in 100% DMSO; 10µM final concentration) into Port A of the Sensor Cartridge (part of a XF96 FluxPak) for later injection into wells containing sample.
  3. Add 25µl of 500mM Sodium Azide (from a 500mM stock dissolved in filter-sterilisedMilliQ water; 50mM final concentration) into Port B of the Sensor Cartridge for later injection into wells containing sample.
  4. Add 25µl of M9 buffer into Ports A and 25µl MilliQ water into Ports B of the Sensor Cartridge for later injection into wells for background correction or blank wells.
  5. Load the Sensor Cartridge and Utility Plate into the Extracellular Flux Analyser.
  6. Allow the machine to calibrate.
  7. Transfer 10 nematodes using a worm-pick into each sample well of a XF96 Cell Culture Microplate containing 200µl M9 buffer.
  8. Once the Extracellular Flux Analyser has completed calibration and is ready to use, remove the Utility Plate, and load the Cell Culture Microplate into the machine and begin the assay.
  9. To obtain data for each key mitochondrial parameter (basal respiration, spare respiratory capacity, and maximal respiration), 6 cycles of OCR measurements are performed, both prior to and after serial addition of FCCP and Azide. Each cycle consists of a Mixing time of 3 minutes, followed by a Waiting time of 2 minutes, and Measuring time of 3 minutes.
  10. The above mitochondrial parameters may be calculated from such data in several ways. One way is to obtain the averages of the OCRs at the various stages, and another is to use the highest (peak) OCR for each particular phase of the experiment. We prefer to use the area under the curve of oxygen consumption rate (AUC OCR).

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