An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes

Published: April 30, 2024

Abstract

Source: Fang, E. F., et al. In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice. J. Vis. Exp. (2017).

This video demonstrates an in vivo assay to study mitophagy in Caenorhabditis elegans. Upon exposing the nematodes to a mitophagy-inducing drug, the occurrence of mitophagy is assessed by co-localization between DCT-1, a mitophagy receptor on the damaged mitochondria, and the autophagosomal membrane protein LGG-1.

Protocol

1. Detection of Mitophagy in C. elegans NOTE: The nematode C. elegans provides a platform to assay mitophagy at the organismal level. Two strains can be used to monitor mitophagy: (1) mitochondria-targeted Rosella (mtRosella) or (2) mitophagy receptor DCT-1 fused with GFP along with autophagosomal marker LGG-1 fused with Discosoma sp. red fluorescent protein (DsRed). Mitophagy monitoring using Rosella biosensor<br…

開示

The authors have nothing to disclose.

Materials

Eclipse TE-2000e confocal microscope Nikon #TE-2000e
Colocalization software Volocity #Volocity 6.3 alternative Zeiss ZEN 2012 software
IN Cell Investigator Software GE Healthcare Life Sciences #28408974
cell culture medium Thermofisher #DMEM–Dulbecco's Modified Eagle Medium
Penicillin-Streptomycin (10,000 U/mL) Thermofisher #15140122
Fetal Bovine Serum Sigma-Aldrich #12003C-1000ML
Cell culture Incubator Thermofisher #Thermo Forma 3110 CO2 Water Jacketed Incubator
epifluorescence microscope Zeiss Zeiss Axio Imager Z2
camera Olympus Olympus DP71
confocal microscope Zeiss Zeiss Axio Observer Z1
confocal software Zeiss ZEN 2012
image analysis software Image J colocalization analysis, etc https://imagej.nih.gov/ij/
statistical analysis software GraphPad Software Inc., San Diego, USA GraphPad Prism software package
material to make a worm pick Surepure Chemetals #4655 The pick is made of 30 gauge 90% platinum 10% iridium wire
IR: N2;Ex[pmyo-3 TOMM-20::Rosella] Material inquiry to Tavernarakis Nektarios Maintain transgenic animals at 20 °C
IR: N2; Ex[pdct-1 DCT-1::GFP; pmyo-3 DsRed::LGG-1] Material inquiry to Tavernarakis Nektarios
pmyo-3 TOMM-20::Rosella Material inquiry to Tavernarakis Nektarios
pdct-1 DCT-1::GFP Material inquiry to Tavernarakis Nektarios
pmyo-3 DsRed::LGG-1 Material inquiry to Tavernarakis Nektarios
Paraquat solution
M9 buffer
M9-levamisole buffer
Glass Microscope Slides and Coverslips Fisher Scientific #B9992000
Surgical forceps STERIS Animal Health 19 Piece Canine Spay Pack Economy
Surgical scissors STERIS Animal Health 19 Piece Canine Spay Pack Economy
1x PBS Thermofisher #AM9625 10x PBS needs to be diluted to 1x PBS by using ddH2O
shaker Fisher Scientific #11-676-178 Thermo Scientific MaxQ HP Tabletop Orbital Small Open Air Platform Shaker Package A
2% agarose pad
Vibroslice blades World precision instruments #BLADES-2 single-edge blade
metal plate MSC #78803988 0.012 in thick x 6 in wide x 12 in long, 430 Stainless Steel Sheet
Triton X-100 detergent
Methyl viologen dichloride hydrate Sigma-Aldrich #856177 paraquat
Incubator for nematodes AQUALYTIC Incubator to maintain 20 °C
Dissecting stereomicroscope Olympus SMZ645
Confocal microscope Zeiss AxioObserver Z1 For nematodes (step 2)
epifluorescence microscope Zeiss AxioImager Z2 For nematodes (step 2)
UV crosslinker Vilber Lourmat BIO-LINK – BLX-E365 UV light source; 356 nm

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記事を引用
An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes. J. Vis. Exp. (Pending Publication), e22133, doi: (2024).

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