Total Triglyceride Quantification in Caenorhabditis elegans

Materials and Reagents

1. 1 L and 2 L Conical flasks (ThermoFisher Scientific, FisherBrand)

2. 60 mm Petri dish with triple vent (Tarsons, catalog number: 460061 )

3. 96-well flat bottom black fluorescent reading plates (SPL Life sciences, catalog number: 30496 )

4. 50 ml graduated centrifuge tube (Tarsons, catalog number: 546021 )

5. 15 ml graduated centrifuge tube (Tarsons, catalog number: 566041 )

6. 0.45 µm filters (GE Healthcare, Whatman^TM, catalog number: 9913-2504 )

7. 0.010-inch diameter Platinum wire

8. Aluminum foil (PRS associates, MYFOIL)

9. 1.5 ml microfuge tube (Trasons)

10. Caenorhabditis elegans

11. Escherichia coli OP50 (Glycerol stock)

12. NaCl (ThermoFisher Scientific, Qualigens, catalog number: 7647 )

13. MgSO₄ (ThermoFisher Scientific, SQ, catalog number: 10034 )

14. CaCl₂ (Merk Life Science, EMPLURA, catalog number: QE5Q651429 )

15. K₂HPO₄ (Avantor^TM, RANKEM, catalog number: P2607 )

16. KH₂PO₄ (ThermoFisher Scientific, Qualigens, catalog number: 7778 )

17. Na₂HPO₄ (ThermoFisher Scientific, Qualigens, catalog number: 7558 )

18. NaOH (SDFCL, catalog number: 20252 K05 )

19. Sodium hypochlorite solution (NaOCl) (Sigma-Aldrich, catalog number: 239305 )

20. Absolute ethanol (Merck Life Science, EMSURE, catalog number: 64-17-5 )

21. Molecular grade water (SIGMA, catalog number: W4502 )

22. Agar powder (HiMedia, catalog number: RM301 )

23. LB broth (BD Biosciences, Difco^TM LB Broth Miller (Luria-Bertani), catalog number: 244620 )

24. LB agar (BD Biosciences, Bacto^TM Agar, catalog number: 214010 )

25. Peptone (BD Biosciences, Bacto^TM Peptone, catalog number: 211677 )

26. Cholesterol (SIGMA, catalog number: C8667 )

27. Streptomycin (Alfa Aesar, catalog number: J61299 )

28. NP-40 solution (SIGMA, catalog number: 74385 )

29. BioVision triglyceride quantification colorimetric/fluorometric kit (BioVision, catalog number: K622 )

    Reagents provided in the BioVision kit

    Triglyceride Assay Buffer (TAB)

    Triglyceride Probe

    Lipase

    Triglyceride Enzyme Mix

    Triglyceride Standard (1 mM)

30. Nematode growth media (see Recipes)

31. 1 M KPO₄ (see Recipes)

32. 1 M CaCl₂ (see Recipes)

33. 1 M MgSO₄ (see Recipes)

34. 5 mg/ml Cholesterol (see Recipes)

35. 50 mg/ml Streptomycin (see Recipes)

36. 5 N NaOH (see Recipes)

37. LB media (see Recipes)

Equipment

1. Water bath

2. Autoclave (JSR, model: JSAX-80 )

3. Laminar flow (JSR, model: JSCB-1200SB )

4. BOD incubator (JSR, model: JSBI-840C )

5. Rocker (SHALOM, GEL ROCKER, model: SLM-GR-100 )

6. Centrifuge (Hettich, Universal 320 R, 1406 )

7. Centrifuge (Eppendorf, Centrifuge 5424 R )

8. Sonicator (BRANSON, DIGITAL SONIFIER 450 )

9. Spectrophotometer (TECAN, model: Infinite M200 pro )

Software

1. i-control^TM (TECAN, https://lifesciences.tecan.com/plate_readers/infinite_200_pro?p=Software)

2. GraphPad Prism5 (PRISM, https://www.graphpad.com/scientific-software/prism/)

Procedure

1. C. elegans maintenance

   1. Streak E. coli OP50 on LB agar plates without any antibiotic (see Recipes) and incubate the plate for 12 h at 37 °C.

   2. Inoculate a single OP50 colony into 10 ml of LB media in a 50 ml graduated centrifuge tube and let it grow overnight (10-12 h) at 37 °C.

   3. Spot ~150 µl of overnight grown OP50 culture to 60 mm NGM plates and dry in laminar flow for ~30 min. These plates can be stored at room temperature (RT).

   4. Using a heat sterilized platinum wire pick, transfer 5-6 gravid worms (Figure 2A) to an OP50 seeded NGM plate and propagate them at 20 °C till they reach the adult stage and start laying eggs.

   5. Adults worms can again be transferred on to a new NGM OP50 plate for maintenance.

      
      

      
      Figure 2. Image of a (A) gravid adult, (B) young adult, and (C) eggs of C. elegans

      
      

2. Lipid quantification

   1. Preparation of synchronized population by taking ~10 gravid adults (60-65 h old worms at 20 °C, see Figure 2A) and allowing them to lay eggs for 6-8 h on NGM plates seeded with OP50. This yields about 500 eggs per plate. Incubate these plates at 20 °C till worms reach the gravid adult stage.

   2. Collect the gravid worms from 3 dishes into 6 ml M9 buffer in a 15 ml graduated centrifuge tube.

   3. Let the worms settle down for 10 min.

   4. Remove excess M9 buffer without disturbing the pellet of worms and add 10 ml autoclaved water.

   5. Let the worms settle down and then remove water up to 2 ml mark in the 15 ml graduated centrifuge tube.

   6. Prepare a 2x bleach mix by adding 5 N NaOH and NaOCl (bleach) in water (see Recipes).

   7. Add 2 ml of 2x bleach mix to the suspension of worms in the water.

   8. Vortex vigorously for 30 s to break open the worms. Check under a microscope to ascertain the release of eggs in the solution (Figure 2).

      Note: The duration of vortexing can vary with the strength of the bleach. Vortex vigorously till worms break open and eggs are released in the solution.

   9. Add 15 ml M9 buffer immediately after eggs are released.

   10. Centrifuge at 850 x g for 3 min for eggs to settle down at the bottom. Remove excess M9 buffer without disturbing the pellet.

   11. Repeat Steps B9 and B10 twice.

   12. Resuspend the eggs in 1 ml of M9 buffer in the graduated centrifuge tube. Incubate on a tube rotator at 20 °C for 18-20 h.

   13. Examine the egg suspension for hatching after 18-20 h.

   14. Shake the tubes properly and spot three 5 µl drops on a plain NGM plate. Count the number of worms in each spot. Take the average of three reads to estimate total numbers of worms present.

   15. Spot hatched L1 larvae on five NGM OP50 plates with an average of 300-400 worms on each and propagate the worms at 20 °C till the young adult stage (45-48 h after spotting L1, see Figure 2).

   16. Collect the worms in a 15 ml graduated centrifuge tube using M9 buffer, wash twice with 10 ml M9 buffer (samples to be on ice till Step B23).

   17. Let the worms settle and remove excess M9 buffer.

   18. Resuspend worms in 15 ml of 5% NP-40 solution in water and let worms settle again.

   19. Remove excess NP-40 solution and resuspend worms in 2 ml of 5% NP-40 solution.

   20. Spot 50 µl of the suspension of worms and count the number of worms. Dilute, if necessary, to keep the same average number (~40-50 worms per 50 µl) of worms across different samples.

   21. Let the worms settle again on ice and resuspend in 500 µl of 5% NP-40 solution.

   22. Sonicate the worms in 5% NP-40 solution on ice for 30 min with 15 s ON and 10 s OFF at 60% amplitude. Change the ice, if required, every 10 min.

   23. Transfer the sonicated lysate to a fresh 1.5 ml microfuge tube.

   24. Heat the sample to 100 °C in a water bath for 2-5 min and then let it cool at RT temperature. This is important to completely solubilize all the triglycerides in the samples.

   25. Repeat Step B24 once.

   26. Centrifuge at 4 °C for 2 min at 12,130 x g.

   27. Transfer the supernatant to a fresh 1.5 ml microfuge tube without disturbing the pellet. Keep the supernatant on ice and discard the pellet.

   28. Make 1:100 dilution of the sample in water for measurement.

       
       

3. Generation of a standard curve for triglycerides

   1. Heat the triglyceride standard, provided in the BioVision kit, to 100 °C in a water bath for 1 min and vortex for 1 min.

   2. Repeat Step C1 twice and let it cool. Make sure the solution is clear.

   3. Dilute the 1 mM triglyceride standard to 0.4 µM (0.4 pmol/µl) with triglyceride assay buffer (TAB).

   4. Prepare buffer control (TAB alone) along with 1:5, 2:5, and 4:1 dilutions of 0.4 µM triglyceride standard with TAB to generate 0, 4, 8, and 16 picomoles triglyceride per well for fluorometric quantification of triglycerides in a 96-well microtiter plate. Adjust volume to 50 µl with TAB.
      Note: For creating “0, 4, 8 and 16 pmol triglyceride”, we added 0, 10, 20, and 40 µl of 0.4 µM standard to 50, 40, 30, and 10 µl of TAB buffer respectively. For technical duplicates, a total reaction volume of 100 µl should be prepared for each standard in 1.5 ml microcentrifuge tubes.

   5. Add 2 µl lipase to each 50 µl of standard and mix well. For duplicates, add 4 µl of lipase to each 100 µl standard, mix well, and split 50 µl in each well of the 96-well microtiter plate. Incubate the mix at room RT on a rocker for 20 min.

      Note: Flat bottom black 96-well microtiter plate is used for this experiment.

   6. Prepare the TAG reaction mix by mixing 2 µl TAG enzyme mix, 0.4 µl TAG probe, and 47.6 µl of TAB for each well. Prepare a master mix for the total number of standard wells plus one buffer reaction to reduce variation. For example, for 4 standards (including buffer control) in duplicates, it will be 18 µl TAG enzyme mix, 3.6 µl TAG probe, and 428.4 µl of TAB buffer. A buffer reaction is added to take care of volume loss due to pipetting errors.

      Note: Protect the reaction from light.

   7. Add 50 µl of the TAG reaction mix to buffer control and each standard well of the 96-well microtiter plate. Incubate the reaction on the rocker for 60 min at RT.

   8. Measure the fluorescence at Ex/Em of 535/590 nm using TECAN Infinite M200 pro spectrophotometer (see Figure 3).

      
      

      
      Figure 3. Steps for generation of triglyceride standard curve using Biovision kit for triglyceride quantification

      
      

   9. Generate standard curve by plotting fluorescence vs triglycerides concentration (pmol) using PRISM (see steps in Figure 3). Obtain a fit by applying linear regression.

   10. The standard curve is used to interpolate values of x (triglyceride concentrations) for known values of y (follow steps in Figure 4).

       
       

       
       Figure 4. Interpolation from the standard curve to obtain triglyceride concentration in biological samples

       
       

4. Measurement of triglycerides in C. elegans samples

   1. Dilute worm supernatant (from Step B28) to 1:2, 1:5, and 1:10 with TAB to make up the final volume to 50 µl in a 96-well microtiter plate, in duplicates.

   2. Add 2 µl lipase to each sample and mix it well. Incubate the mix at RT on a rocker for 20 min.

   3. Include no lipase control for each sample to measure basal level glycerol present in the sample.

   4. For each well, prepare 50 µl of triglyceride reaction mix by adding 2 µl Triglyceride enzyme mix, 0.4 µl triglyceride probe, and 47.6 µl TAB. Prepare a single master mix for all the wells.

      Note: Protect the reaction from light.

   5. Add 50 µl of the triglyceride reaction mix to each sample well of the 96-well microtiter plate (from Step D2) and incubate at RT for 60 min.

   6. Measure the fluorescence at Ex/Em of 535/590 nm for each sample with lipase and respective no lipase control (raw values shown in the snapshot in Figure 5).

      
      

      
      Figure 5. Calculations for triglyceride quantification in C. elegans samples

Data analysis

1. Subtract the fluorescence value of no lipase control from fluorescence value of respective worm sample with lipase. This provides corrected fluorescence value.

2. Apply the corrected fluorescence values to the triglyceride linear regression fit line (from Step C9) to get the picomoles of TAGs present in sample per well by interpolation in GraphPad Prism5 (see Figure 4 and Figure 5).

3. Calculate the concentration of triglycerides per µl in worm samples (Figure 5).

   
   
   
   

   where,

   Z: picomoles of triglycerides per µl of the sample

   a: volume of sample added to the well

   d: sample dilution factor

4. Absolute amount of triglycerides present per worm can be calculated (Figure 5)

   
   
   
   

   where,

   Q: picomoles of triglycerides per worm

   w: number of worms per present ul of the sample

   
   
   
   

   where,

   n: total number of worms for each sample (from Step B20)

   r: volume of worm suspension in Step B19

   
   

   Here, we have shown an example of TAGs quantification in wild type N2 animals (denoted as WT) and str-2 (ok3148) animals. STR-2 chemosensory G-protein coupled receptor controls the life span of C. elegans at high temperatures by regulating fat metabolism. str-2 mutants have reduced total lipid content as confirmed by Oil-Red-O staining and triglycerides quantification by BioVision kit (Figure 6). For WT and str-2 (ok3148) animals, we found that the triglyceride level was 0.78 ± 0.03 and 0.58 ± 0.03 nanomoles per worm respectively (Dixit et al., 2020). One of the caveat of the biochemical method is that it can not provide information on the spatial distribution of stored fats in various tissues of worms. Thus, a combination of staining methods and biochemistry is desirable for the study of fat metabolism in worms.

   
   

   
   Figure 6. Triglycerides levels in WT and str-2 (ok3148) mutant of C. elegans. A. Mean ± SEM triglycerides/worm in WT and str-2 animals. B. Lipids droplets stained by Oil-Red-O in WT and str-2 animals (Scale bar: 200 µm).

Recipes

1. Nematode growth media (1 L)

   3 g NaCl

   2.5 g Peptone

   17 g Agar

   Water up to 1 L

   Autoclave the medium at 121 °C and 15 psi for 30 min

   Cool down the media to ~50 °C and add the following: 

   1 ml 1 M CaCl₂

   1 ml 1 M MgSO₄

   1 ml of 5 mg/ml Cholesterol

   1 ml 50 mg/ml streptomycin

   25 ml 1 M KPO₄

   Mix well and pour in 60 mm Petri dish

2. 1 M KPO₄ (200 ml)

   21.66 g of KH₂PO₄

   7.12 g of K₂HPO₄

   Water up to 200 ml

   Autoclave the preparation at 121 °C and 15 psi for 30 min

3. 1 M CaCl₂ (100 ml)

   14.7 g of CaCl₂

   Water up to 100 ml

   Autoclave the solution at 121 °C and 15 psi for 30 min

4. 1 M MgSO₄ (100 ml)

   12.037 g of MgSO₄

   Water of 100 ml

   Autoclave the solution at 121 °C and 15 psi for 30 min

5. 5 mg/ml Cholesterol

   0.25 g of cholesterol

   50 ml absolute ethanol

   Filter sterilize the solution with 0.45 µm filter

6. 50 mg/ml Streptomycin

   0.5 g of streptomycin

   Water up to 10 ml

   Filter sterilize with 0.45 µm syringe filter

7. 5 N NaOH

   8 g NaOH

   Water up to 40 ml

   Filter sterilize with 0.45 µm syringe filter

8. LB media

   2.5 g LB broth

   water up to 100 ml

   Autoclave the LB broth at 121 °C and 15 psi for 30 min

9. LB agar

   2.5 g LB broth

   2 g agar

   Water up to 100 ml

   Autoclave the preparation at 121 °C and 15 psi for 30 min

   Cool down the media to ~50 °C and pour in 60 mm Petri dishes

10. M9 buffer (500 ml)

    1.5 g KH₂PO₄

    3 g Na₂HPO₄

    2.5 g NaCl

    0.5 ml of 1 M MgSO₄

    Water up to 500 ml

    Autoclave the solution at 121 °C and 15 psi for 30 min

11. 2x Bleach Mix (2 ml)

    400 µl 5 N NaOH

    900 µl of NaOCl (bleach)

    Water up to 2 ml

Acknowledgments

This work was supported by the Wellcome Trust/DBT India Alliance Fellowship (Grant no. IA/I/13/1/500919) awarded to Varsha Singh.; 2) This protocol was used in our original study in Dixit et al. (2020).

Competing interests

We declare no financial or non-financial competing interests.

References

1. Ashrafi, K., (2007). Obesity and the regulation of fat metabolism. WormBook 1-20.
2. Ashrafi, K., Chang, F. Y., Watts, J. L., Fraser, A. G., Kamath, R. S., Ahringer, J. and Ruvkun, G. (2003). Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421(6920): 268-272.
3. Cameron, J. L., Capuzzi, D. M., Zuidema, G. D. and Margolis, S. (1974). Acute pancreatitis with hyperlipemia. Evidence for a persistent defect in lipid metabolism. Am J Med 56(4): 482-487.
4. Cohen, S., (2018). Lipid Droplets as Organelles. Int Rev Cell Mol Biol 33783-110.
5. Dixit, A., Sandhu, A., Modi, S., Shashikanth, M., Koushika, S., Watts, JL and Singh, V. (2020). Neuronal control of lipid metabolism by STR-2 G protein coupled receptor promotes longevity in. Caenorhabditis elegans. AGING CELL 19: e13160.
6. Kimura, K. D., Tissenbaum, H. A., Liu, Y. and Ruvkun, G. (1997). daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277(5328): 942-946.
7. Lee, C. H., Olson, P. and Evans, R. M. (2003). Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 144(6): 2201-2207.
8. Mak, H. Y., (2012). Thematic review series: Lipid droplet synthesis and metabolism: From yeast to man: Lipid droplets as fat storage organelles in Caenorhabditis elegans. J Lipid Res 53(1): 28.
9. Onal, G., Kutlu, O., Gozuacik, D. and Emre, S. D. (2017). Lipid droplets in health and disease. Lipids Health Dis 16(1): 1-15.
10. O'Rourke, E. J., Soukas, A. A., Carr, C. E. and Ruvkun, G. (2009). C. elegans major fats are stored in vesicles distinct from lysosome-related organelles. Cell Metab 10(5): 430-435.
11. Stobart, A. K., Stymne, S. and Höglund, S. (1986). Safflower microsomes catalyse oil accumulation in vitro: a model system. Planta 169(1): 33-37.
12. Suzuki, M., Shinohara, Y., Ohsaki, Y. and Fujimoto, T. (2011). Lipid droplets: size matters. J Electron Microsc (1): S101-S116.
13. Walther, T. C. and Farese Jr, R. V. (2012). Lipid droplets and cellular lipid metabolism. Annu Rev Biochem 81: 687-714.
14. Yen, K., Le, T. T., Bansal, A., Narasimhan, S. D., Cheng, J. X. and Tissenbaum, H. A. (2010). A comparative study of fat storage quantitation in nematode Caenorhabditis elegans using label and label-free methods. PLOS ONE 5(9): e12810.