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 |
