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Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.
Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.
C. elegans are a powerful model organism for research in genetics, cellular biology, and molecular biology, because they are easily cultured in the laboratory, have a short generation time and lifespan, share a high degree of protein homology with mammals, and have a transparent body structure that allows in vivo visualization of fluorescent proteins and dyes1. As a result of the long-standing use of C. elegans as a major model system in a range of fields, including developmental biology and aging, their growth and development are well-understood, their genome has been fully sequenced, and a host of powerful genetic t....
1. Recipes
NOTE: Prepare stock solutions before starting microtray plate preparation.
The microtray-based single-worm culture environment described here can be used to monitor a variety of phenotypes, including lifespan and health span, activity and movement, body shape and crawling geometry, and the expression of transgenically expressed fluorescent biomarkers in individual animals over time. The microtray culture system is compatible with lifespan analysis through either manual scoring or image collection and downstream imaging analysis. As with standard culture on Petri plates21.......
Here, we describe a novel culture system that adapts microtrays, originally developed for human leukocyte antigen tissue typing assays, to allow the isolation and characterization of single C. elegans over time in a solid media environment that is contextually similar to the agar-based NGM that is the standard in C. elegans research. This system is compatible with a variety of interventions, including dietary restriction, exogenous drug treatment, a challenge with chemical or environmental stressors, an.......
This work was supported by NIH R35GM133588 to G.L.S., an NIHT32GM008659 training grant to L.E., a United States National Academy of Medicine Catalyst Award to G.L.S., and the State of Arizona Technology and Research Initiative Fund administered by the Arizona Board of Regents.
....Name | Company | Catalog Number | Comments |
3D-printed terasaki inserts | Custom printing company | Robot_Terasaki_tray_insert_10-20 -2021.STL | FDM printing, nozzle size 0.6 mm using standard PLA plus filament |
AirClean systems AC624LF vertical laminar flow fume hood | Fisher Scientific | 36-100-4376 | |
Bacto peptone | Thermo Scientific | 211677 | |
CaCl2 | Acros organics | 349615000 | |
Caenorhabditis elegans N2 | Caenorhabditis Genetics Center (CGC) | N2 | Wildtype strain |
Carbenicillin | Goldbio | C-103-25 | |
Cholesterol | ICN Biomedicals Inc | 101380 | |
Escherichia coli OP50Â | Caenorhabditis Genetics Center (CGC) | OP50 | Standard labratory food for C. elegans |
Ethanol | Millipore | ex0276-4 | |
Fisher Vortex Genie 2 | Fisher Scientific | G-560 | |
FUdRÂ | Research Products International | F10705-1.0 | |
Hydrating water crystals | M2 Polymer Technologies | Type S | Type S super absorbent polymer |
Isopropyl ß-D-1-thiogalactopyranoside (IPTG) | GoldBio | I2481C100 | |
K2HPO4 | Fisher Chemical | P288-500 | |
Kimwipes | KimTech | 34155 | Task wipes |
LB Broth, Lennox | BD Difco | 240230 | |
Leica K5 sCMOS monochrome camera | Leica Microsystems | 11547112 | |
Leica M205 FCA Fluorescent Stereo Microscope | Leica Microsystems | 10450826 | |
Low-melt agarose | Research Products International | A20070-250.0 | |
MgSO4Â | Fisher Chemical | M-8900 | |
NaCl | Fisher bioreagents | BP358-1 | |
Nunc OmniTray Single-Well Plate | Thermo Scientific | 264728 | |
Nystatin | Sigma | N1538 | |
Palmitic acid | Acros organics | 129700010 | |
Paper towels | Coastwide Professional | 365374 | |
Parafilm M | Parafilm | 16-101 | |
Stratagene UV Stratalinker 2400 | Stratagene | 400075 | UV crosslinker |
Terasaki trays (Lambda) | One Lambda | 151431 | |
Thermolyne Dri-bath | Thermolyne | DB28125 | |
Tween | Thermo Scientific | J20605-AP |
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