Protocol for assessing the healthspan of Caenorhabditis elegans after potential anti-aging drug treatment

Summary

The nematode Caenorhabditis elegans has been developed as a valuable genetic model for research on aging and aging-related diseases. Here, we present a protocol for assessing the healthspan of C. elegans after treatment of a potential anti-aging drug. We describe steps for C. elegans synchronization, drug treatment, and lifespan determination from the survivorship curve. We also detail assessment of locomotory ability through body bend rate and measurement of lipofuscin fluorescence to quantify age pigment in the worm intestine.

For complete details on the use and execution of this protocol, please refer to Xiao et al. (2022).¹

Graphical abstract

Highlights

• •Protocol to test the effect of a potential anti-aging drug on the lifespan of C. elegans
• •Quantification of age pigment by measuring lipofuscin fluorescence in the worm intestine
• •Assessment of locomotory ability through body bend rate
• •Applicable for testing other potential anti-aging drugs in C. elegans

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The nematode Caenorhabditis elegans has been developed as a valuable genetic model for research on aging and aging-related diseases. Here, we present a protocol for assessing the healthspan of C. elegans after treatment of a potential anti-aging drug. We describe steps for C. elegans synchronization, drug treatment, and lifespan determination from the survivorship curve. We also detail assessment of locomotory ability through body bend rate and measurement of lipofuscin fluorescence to quantify age pigment in the worm intestine.

Before you begin

Worms were maintained and propagated under standard conditions.^2,3

Institutional permissions

Experiments involving nematodes C. elegans do not require permission from Institutional Animal Care and Use Committee(IACUC).

Media preparations

Timing: 1 week

In the next section, we will prepare the media and buffers, which are necessary to assess lifespan, body bends and age pigment.

• 1.Preparation and preservation of the potential anti-aging drug: Usnic acid is obtained from Sigma Chemical Co. (St. Louis, MO), and dissolved in Dimethyl sulfoxide (DMSO) as a stock solution at a 100 mM concentration and is stored in aliquots at −20°C.

Note: The dissolved Usnic acid can be stored at −20°C for up to three months. In addition, the final concentration of DMSO should be less than 0.5% in experiments.

• 2.Prepare lysogeny broth (LB) agar plates and liquid medium. Autoclave the medium for 20 min at 121°C.
• 3.
  Culturing of Escherichia coli (E. coli) strain OP50.

  • a.
    Streak E. coli OP50 on a LB agar plate from a glycerol stock and incubate the bacteria streak at 37°C for 16 h.
  • b.
    Inoculate 100 mL conical flask containing 50 mL of sterile LB with E. coli OP50 from the streak plate.
  • c.
    Incubate the broth at 37°C for 16–24 h in a shaking incubator maintained at 180 rpm.

Note: The E. coli OP50 streak plate with wrapping in parafilm can be stored at 4°C for up to ten days. In addition, the E. coli OP50 fresh liquid culture can be stored at 4°C for up to three days.

• 4.
  Prepare Nematode Growth Medium (NGM).

  • a.
    Autoclave the medium for 20 min at 121°C.
  • b.
    Allow the medium to cool to 50°C in a water bath.
  • c.
    Add 1 mol/L MgSO₄ (1 mL), 1 mol/L phosphate buffer (K₂HPO₄/KH₂PO₄) (25 mL), 1 mol/L CaCl₂ (1 mL), and 5 mg/mL Cholesterol (dissolved in 100% ethanol) (1 mL).
  • d.
    Filter sterilizes the above solution through a 0.22 μm filter.

    Note: In general, each 60 mm petri dish requires approximately 10 mL NGM to fill (used in worms culture). Each 35 mm petri dish requires approximately 4 mL to fill (used in healthspan assay).

  • e.
    Let NGM plates cool on bench 16 h at 20°C.

    Note: 1 mol/L MgSO₄, 1 mol/L phosphate buffer (K₂HPO₄/KH₂PO₄), 1 mol/L CaCl₂, and 5 mg/mL Cholesterol can be stored at 4°C for up to three months. The fresh NGM plates can be stored at 4°C for up to 1 month.

    CRITICAL: Do not add the MgSO₄, CaCl₂, Cholesterol, or phosphate buffer (K₂HPO₄/KH₂PO₄) before the NGM cools to 50°C as it will cause the reagents to precipitate out of the medium and form crystals.

• 5.Seed the fresh 60 mm NGM plates with 500 μL of the fresh E. coli OP50 liquid culture and spread the liquid culture by gently swirling the plate. Seed the fresh 35 mm NGM plates with 250 μL of the fresh E. coli OP50 liquid culture and spread the liquid culture by gently swirling the plate. Allow the fresh E. coli OP50 liquid dry on super clean workbench.

Note: The seeded NGM plates can be stored at 20°C for up to two weeks.

• 6.Prepare M9 buffer. M9 buffer (1L) containing NaCl (5 g), 1 mol/L MgSO₄ (1 mL), KH₂PO₄ (3 g), Na₂HPO₄ (6 g), and add ddH₂O up to the volume of 1 L. Filter sterilizes the above solution through a 0.22 μm filter.

Note: The M9 buffer can be stored at 20°C for up to three months.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
E. coli OP50	Yun Nan University	N/A
Chemicals, peptides, and recombinant proteins
Usnic acid	Sigma-Aldrich	CAS:7562-61-0
Bleach	Xilong Chemical Co	N/A
Agarose	Biowest	CAS: 111860
Peptone	Solarbio	P8450
Tryptone	AOBOX	01-004
Sodium chloride	Solarbio	S8210
Yeast extract powder	Sangon Biotech	A515245-0500
Agar	Biofroxx	8211GR500
Calcium chloride	Sangon Biotech	A501330-0500
Magnesium sulfate	Solarbio	M8301
Potassium dihydrogen phosphate	Sangon Biotech	A501211-0500
Dipotassium phosphate	Sangon Biotech	A501212-0500
Cholesterol	Sangon Biotech	A506234-0025
Absolute ethyl alcohol	ACMEC	E99944
Sodium hydroxide	ACMEC	S41250-500g
Disodium hydrogen phosphate	MACKLIN	S818100
Sodium hypochlorite solution	KESHI	N/A
Experimental models: Organisms/strains
Caenorhabditis elegans wild-type (N2)	Caenorhabditis Genetics Center	N/A
Other
1.5 mL EP(eppendorf) tube	LABSELECT	MCT-001-150
15 mL centrifuge tube	LABSELECT	CT-012-15
Vortex oscillator	Jiangsu Tenlin Instrument Co, LTD	N/A
Microscope slides	SAIL BRAND	CAT.NO.7101
60 mm Petri dish	LABSELECT	12211
35 mm Petri dish	LABSELECT	12111
Autoclave	SHENAN	LDZM-80L-1
Upright metallurgical microscope	Nexcope	HE610
Stereo microscope	Mshot	MZ62
Fluorescence microscope	OLYMPUS	IX97
Biochemical incubator	Shanghai Boxun	SPX-250B-Z
Zeiss Imager M2 microscope	Zeiss	Imager M2
Software and algorithms
GraphPad Prism	GraphPad	https://www.graphpad.com/scientific-software/prism/ RRID: SCR_002798
ImageJ	Schneider et al.4	https://imagej.nih.gov/ij/

Materials and equipment

1 L of LB medium

Reagent	Amount
NaCl	10 g
Tryptone	10 g
Yeast Extract	5 g
Agar	17 g
ddH2O	1L

1 L of NGM

Reagent	Final concentration	Amount
NaCl	51 mM	3 g
Peptone	N/A	2.5 g
Agar	N/A	17 g
ddH2O	N/A	975 mL
MgSO4 (1 M)	1 mM	1 mL
Phosphate buffer (1 M)	25 mM	25 mL
CaCl2 (1 M)	1 mM	1 mL
Cholesterol (5mg/mL)	5 μg/mL	1 mL

1 L of M9 buffer

Reagent	Final concentration	Amount
NaCl	85 mM	5 g
MgSO4 (1 M)	1 mM	1 mL
KH2PO4	22 mM	3 g
Na2HPO4	42 mM	6 g
ddH2O	N/A	1L

Step-by-step method details

Experiment preparations

Timing: 1 week

In this section, we describe how to synchronize wild-type C. elegans and prepare E. coli OP50 NGM plates with or without a potential anti-aging drug for measuring healthspan.

• 1.Maintain wild-type worms in NGM plates seeded with E. coli OP50 until gravid adult at 20°C (2–3 days).

Note: Avoid nematodes starvation, also be careful of overcrowding the plates by transferring them to fresh plates seeded with E. coli OP50 every 2–3 days.

• 2.
  Remove bacterial or yeast contaminants from C. elegans stock plates and obtain a synchronized population of worms for experiments.³

  • a.
    Use C. elegans stock plates that have many gravid hermaphrodites. Wash the plates with sterile M9. Pipet the M9 across the plate several times to loosen worms and eggs that are stuck in the bacteria.
  • b.
    Collect the liquid in a sterile 15 mL conical centrifuge tube with cap. Let worms settle (approx 5 min) and remove most of the water carefully, so as not to disturb the loose worm pellet.
  • c.
    For preparing eggs from each strain, make the lysis solution by mixing 0.3 mL 5 M NaOH with 1.2 mL bleach and 3.5 mL H₂O. Add the solution to the centrifuge tube with the worms.

    CRITICAL: Make this solution fresh just before use!

  • d.
    Rock the tube gently back and forth for about 3–5 min.

    • i.
      Check periodically, at first you will see intact worms, eventually they will start to dissolve.
    • ii.
      When most of worms have dissolved, give the tube a good shake or vortex. This will dissolve the remaining corpses.

      CRITICAL: Be sure not to leave the worms in the bleach solution any longer than necessary to dissolve the worms. If the eggs are exposed to bleach for too long they will die.

  • e.
    Spin the tube in a desktop centrifuge for 1 min at 1,300 × g to pellet released eggs.
  • f.
    Aspirate the supernatant and leave about 0.1 mL solution in the tube. Add sterile M9 to 12 mL. Shake well or vortex for a few seconds.
  • g.
    Repeat steps e and f.
  • h.
    Transfer the precipitation with 5–10 mL sterile M9 to a clean petri dish.
  • i.
    Place the plate in a constant temperature incubator at 20°C. After about 16 h, the eggs of the worm could be incubated into L1 or L2.

    Note: The lysis time of nematodes should not exceed 5 min.

    Note: The lysate should be cleaned, or the eggs may not hatch.

• 3.Transfer synchronous nematodes to NGM plates seeded with E. coli OP50 until they reach L4 stage, which takes 48 h at 20°C.

Note: Avoid nematodes starvation by transferring them to fresh plates seeded with E. coli OP50.

• 4.Streak fresh E. coli OP50 on a new LB agar plate on the same day of animal synchronization and incubate the plate at 37°C for 16 h.
• 5.The following day, inoculate 5 mL of liquid LB with a single colony from the E. coli OP50 streak plate, at 180 rpm, 37°C for 16–24 h.
• 6.Seed the fresh 35 mm NGM plates with 250 μL of the fresh E. coli OP50 liquid culture with or without the potential anti-aging drug (2 μM Usnic acid; add the drug to the autoclaved NGM media to mix before pouring the media into the petri dish). Allow the plate with the fresh E. coli OP50 liquid dry on super clean workbench.

Assess the lifespan after drug-treatment in C. elegans

Timing: no less than 20 days

In this section, we describe how to determine the lifespan after drug treatment.

• 7.Transfer 30 synchronized L4 animals to each of the fresh 35 mm NGM plates seeded with E. coli OP50 and with or without drug (2 μM Usnic acid) at 20°C.
• 8.Measure the time from day one to observe the growth of nematodes
• 9.For the first 10–14 days, transfer worms everyday and afterwards every second day.

Note: Contamination should be avoided during the transfer of nematodes.

• 10.Mark nematodes that do not respond to mild stimuli as dead. Animals that climb down from plates or show unnatural deaths, especially due to internal hatching of animals are excluded from analysis. Refer to Figure 1 for lifespan data.

Note: Three plates are tested per assay and all experiments are performed three times independently.

Figure 1.

Usnic acid treatment extended the lifespan in wild-type animals

Kaplan-Meier survival curves of N2 hermaphrodite worms exposed to 2 μM Usnic acid (p < 0.05; log-rank test).

Assess the content of age pigment after drug-treatment in C. elegans

Timing: 10 days

In this section, we describe how to determine the content of age pigment after drug treatment.

• 11.Synchronize nematodes and treat for 10 days with or without drug (2 μM Usnic acid) starting at L4 larvae stage.

Note: Avoid nematodes starvation by transferring them to fresh plates seeded with E. coli OP50 every two days.

• 12.
  Mount worms onto an agarose pad attached to a glass slide.

  • a.
    To mount, we use 2% agarose pads made by dissolving 2% agarose in water in a microwave.
  • b.
    Place 2-3 pieces of tape on two microscope slides and put anon-taped slide in the middle in parallel.
  • c.
    Put 50 μL of the 2% agarose on the middle slide and immediately drop another slide on top perpendicularly, so that the top slide is buffered from hitting the bottom by the tape.
  • d.
    Wait about a minute and remove the top slide, leaving the agarose pad attached to the bottom slide.
• 13.Take images with Zeiss Imager M2 microscope. The microscope magnification is 10 (objective) ×10 (eyepiece). Excitation wave length is 400 nm and detection wave length is 570 nm. Refer to Figure 2 for age pigment.
• 14.
  Measure fluorescence intensity by Image J.⁴

  Note: Worms number (n) ≥30. Refer to Figure 2 for quantification of age pigment.

  • a.
    Install the Image J software, open the software, click File-Open, open a fluorescent image that needs to be processed.
  • b.
    Click the interface image-type-8-bit to get a picture.
  • c.
    Flip image: Edit-Invert.
  • d.
    Image calibration: Click the interface Analyze-Calibrate, select Uncalibrated OD, click OK, and then close the next step. Adjust the threshold and select the appropriate area: click Image-Adjust-Threshold.
  • e.
    Quantitative output parameters are set: Analytic-set measurements, (check Mean gray value and Limit to threshold). Options need to be set according to the experimental requirements.
  • f.
    Output data: Analyze-Measure to record intDen data. excel was used to record and sort out all intDen values, and the sorted data was input into the analysis software for significance analysis and mapping.

    Note: All experiments are performed three times independently.

    Note: The bright color (light blue) in Figure 2 represents age pigment, which can be quantified by measuring lipofuscin fluorescence in worm intestine.

Figure 2.

Usnic acid treatment decreased the content of age pigment in C. elegans

Nematodes were synchronized and treated for 10 days with or without 2 μM Usnic acid starting at L4 larvae stage. “Age pigment” is quantified by measuring lipofuscin fluorescence normalized to auto-fluorescence in 2 μM Usnic acid-treated worms versus the control (n ≥ 30). (∗∗P < 0.01, unpaired t-test). The microscope magnification is 10 (objective)×10 (eyepiece). Excitation wave length is 400 nm and detection wave length is 570 nm. Error bars represent mean ± SEM of 3 independent biological replicates. Scale bar: 50 μm.

Assess the locomotory ability after drug-treatment in C. elegans

Timing: 5 and 10 days

In this section, we describe how to determine the locomotory ability after drug treatment.

• 15.Synchronize nematodes and treat with or without drug (2 μM Usnic acid) starting at L4 larvae stage.

Note: Avoid nematodes starvation by transferring them to fresh plates seeded with E. coli OP50.

• 16.On days 5 and 10 of life from L4 stage, measure 20 individuals from the control and experimental plates for body bend rate in liquid.

Note: We count every time when there is a head swing.

• 17.Place worms in 20 μL M9 buffer on a glass slide and film with a Zeiss Imager M2 microscope.

Note: Worms number (n) ≥20. Refer to Figure 3 for quantification of body bend rate.

Note: Body bends are counted by reviewing each frame of the 60 s film.

Note: All experiments are performed three times independently.

Figure 3.

Usnic acid treatment increased the locomotory ability in C. elegans

Nematodes were synchronized and treated with or without 2 μM Usnic acid starting at L4 larvae stage. On days 5 and 10 of life, 20 individuals from the control and experimental plates were measured for body bend rate in liquid. Locomotion as determined by the number of body bends per min in drug-treated worms versus the control (n ≥ 20). (∗∗P <0.01, unpaired t-test). Error bars represent mean ± SEM of 3 independent biological replicates.

Expected outcomes

Usnic acid treatment extended the lifespan in wild-type animals (Figure 1). Furthermore, Usnic acid treatment decreased the age pigments by measuring lipofuscin fluorescence in C. elegans (Figure 2) and increased the locomotory ability (determined by the average bends of the worm body per 60 s) (Figure 3). Graphs were generated with GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA). Statistical analyses for all data except for lifespan assays were carried out using Student’s t-test (unpaired, two-tailed) or ANOVA after testing for equal distribution of the data and equal variances within the dataset. Survival data were analyzed by using the log rank (Mantel-Cox) test.

Limitations

This protocol describes how to quantify the healthspan of C. elegans after potential anti-aging drug treatment. First, the drug must be completely dissolved, otherwise it will affect the final experimental results. Secondly, due to the long cycle of the lifespan assay, lack of sterile technique could result in contamination with bacteria that are not E. coli and may cause aversion or other behavioral and immune responses during the experiment, resulting in inaccurate experimental results.

Troubleshooting

Problem 1

Drug concentrations are difficult to determine (steps 7, 11, and 15).

Potential solution

In the early stage, we conducted a large number of drug (natural products) screening in lifespan assay, and found that the effective concentration of drugs was generally within (0.1 μM, 1 μM, 10 μM).

Problem 2

Difficulty in dissolving drugs (natural products) (steps 7, 11, and 15).

Potential solution

Normally, sterile water is a good solvent because it doesn’t have adverse effects on organisms, but many drugs (natural products) don’t dissolve in water. Therefore, we will dissolve the drugs (natural products) in dimethylsulfoxide (DMSO). In order to reduce the toxic side effects of DMSO on the organism, we will control the final concentration of DMSO to be less than 0.5% during the experiment. Ensure that there are DMSO controls when required.

Problem 3

Difficulty transferring animals from one plate to another. Regular transfer of worms can induce stress and potentially kill a few worms in the process of transfer (step 9).

Potential solution

The worm pick should be bent to resemble a hockey stick and the end flattened to increase surface area.³

Problem 4

The problem of bacterial contamination during transferring animals in lifespan assay (step 9).

Potential solution

When transferring animals from one plate to another, we will choose a laboratory similar to the cell operation room to carry out, turn on the ultraviolet light for 30 min, and then transfer animals.

Problem 5

What if the drug is toxic to the E. coli.

Potential solution

The heat-killed E. coli OP50 can be used for related experiments.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yun Liu (liuyunzmu@126.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate data or code.