Lifespan Analysis: Measuring C. elegans Longevity

Overview

This video describes a traditional, plate-based method of measuring the lifespan of C. elegans. The example protocol measures the lifespan of worms treated with RNAi.

Protocol

The following protocol is an excerpt from Cornwell et al, The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan, J. Vis. Exp.  (2018).

1. Preventing progeny production by the addition of 5-Fluoro-2'-deoxyuridine (FUdR)

1. Grow animals until L4 stage at 20 °C. Check to see whether synchronized animals have developed to L4 approximately 40 h after seeding (step 2.1.7).
   NOTE: C. elegans developmental time will vary at different temperatures and growth rates of mutant animals for which rates have not been characterized must be empirically tested.
   1. Add 160 µL of 50x FUdR to each 6 cm plate with L4 animals.
      NOTE: It is critical to add FUdR at the L4 stage. For convenience, make 1,000x stocks of FUdR by dissolving 1 g FUdR into 10 mL ultrapure H₂O. Filter-sterilize stock FUdR with a 0.2 µm filter and a 10 cc syringe. Aliquot ~1 mL of stock into sterile 1.5 mL tubes. Freeze and store at -20 °C.
2. Inspect plates for the presence of male C. elegans. Remove all males.
   NOTE: Lifespan is typically measured for hermaphrodites and not males. Hermaphrodites that mate live shorter than unmated animals, even in the presence of FUdR. Males are smaller and thinner than hermaphrodites and can be easily identified by a distinctive hooked tail.
3. Put box back into the zip-lock bag. Return to 20 °C incubator.

2. Scoring viability

1. Score viability daily by gently touching the animal on the head with a platinum wire or eyelash. Score animals that fail to move as dead and remove them from the plate (Figure 1A). Record the number observed dead for each condition for every observation time point.
   NOTE: To reduce the risk of scoring bias, the experimenter must remain blind to experimental conditions and similarly must not reference the results from previous time points during scoring.
2. Censor animals that rupture, die from other obvious development defects, or crawl up on the side of the dish: remove these animals and record the number at each observed time point for consideration in the statistical analysis. Additionally, if males are found, remove them and record the number observed.
3. Repeat from step 2.1 daily until no animals remain alive.
   NOTE: Should the lawn of E. coli diminish significantly or fungus begins to grow upon the plate, transfer all remaining animals to a fresh plate with the appropriate RNAi and FUdR.

Representative Results

Figure 1: The Traditional and the Replica Set Method for scoring C.elegans lifespan (A). The Traditional Method for scoring C. elegans lifespan. Several small synchronized populations of isogenic animals per condition are followed over time. The same population of animals is followed throughout the study course. Viability is assessed by movement, which may be stimulated by gentle prodding. Animals that fail to move are scored as dead and are removed (aspiration shown) until no viable animals remain. (B). The Replica Set Method for scoring C. elegans lifespan. A large population of age-synchronized isogenic animals are distributed across a number of identical replicate plates. At each time point, a single replicate is scored: a mild buffered solution (M9) is added, which stimulates movement. Animals that fail to move spontaneously after flooding wells are also assessed via touch stimulus. The scoring duration for the experiment is determined prior to the start. Each animal is scored only once and longevity for the larger population is derived from many independent observations. (C). The Replica Set approach is a high throughput method to quantitatively measure C. elegans lifespan. 100 or more independent RNAi clones can be tracked simultaneously. HT115 E. coli expressing dsRNA for a given RNAi clone is shown. Practically, every 24 samples from the 96-well plate are divided into a single 24-well plate. Each resulting 24-well plate has a negative (i.e. empty vector, red well) and positive control (green well) randomly distributed within a collection of RNAi clones (yellow wells). Typically, the first well (A1) in a collection contains an empty vector. Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
FuDR (5-Fluoro-2'-deoxyuridine)	Alfa Aesar	L16497
24 Well Culture Plates	Greiner Bio-One	#662102
600 µL 96-well plates	Greiner Bio-One	#786261
2mL 96-well plates	Greiner Bio-One	#780286
96-pin plate replicator	Nunc	250520
C. elegans RNAi clone library in HT115 bacteria- Ahringer	Source Bioscience	C. elegans RNAi Collection (Ahringer)	See also Kamath et. al, Nature 2003.
C. elegans RNAi clone library in HT115 bacteria- Vidal	Source Bioscience	C. elegans ORF-RNAi Resource (Vidal)	See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.
L4440 Empty Vector Plasmid	Addgene	1654	https://www.addgene.org/1654/