Name: assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans
Uploaded: 2018-04-13T14:30:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

We would like to thank the Caenorhabditis Genetics Center for strains, the Natural Sciences and Engineering Research Council (NSERC), and the Canada Foundation for Innovation (CFI) for funding. We would like to thank Dr. Lizhen Chen (Department of Cell Systems and Anatomy, UT Health San Antonio) for allowing unrestricted use of her lab facilities for all C. elegans experiments as well as Dr. Exing Wang (Associate Director, Optical Imaging Facility, UT Health San Antonio) for assistance with confocal microscopy. We would also like to thank Dr. Myron Ignatius for providing support and encouragement to facilitate the video shoot.

1. Stain and Image intestinal lysosomes
<ol>
	<li>Seed nematode growth media (NGM) plates with OP50
	<ol>
		<li>Prepare NGM plates as per the recommended protocol19 and allow the closed plates to dry for 2 days at room temperature.</li>
		<li>Inoculate OP50 bacteria into sterile Luria Broth (LB) broth and grow in a shaking incubator or water bath at 37 &#176;C for 36 h or until the OD is between 0.2 and 0.4. Avoid using a bacterial culture with OD &#62;1 or OD &#60;0.2.</li>
		<li>Once NGM plates are dry, place a drop (~30 &#181;L) of OP50 inoculum onto the center of the plate, and then spread the drop into a patch roughly that is 2 cm x 2 cm in size. 
		NOTE: Any remaining OP50 inoculum can be stored at 4 &#176;C for up to a month.</li>
		<li>Invert the OP50 plates once the inoculum has dried (roughly 10 or 15 min), and then incubate the plates at 37 &#176;C for 36 h.</li>
		<li>After 36 h, remove the plates from the incubator and store at 4 &#176;C until later use. 
		NOTE: The plates should be good for up to 1 month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Supplementing cDCFDA to OP50
	<ol>
		<li>To supplement cDCFDA onto OP50 plates, prepare a working stock of 10 mM cDCFDA in dimethyl sulfoxide (DMSO). Keep the cDCFDA solution protected from light and store at -20 &#176;C for later use.</li>
		<li>Gently place 100 &#181;L of 10 mM cDCFDA solution onto the surface of the 2 cm x 2-cm OP50 patch, such that the entire patch is uniformly covered with cDCFDA. 
		NOTE: It is very important that the entire patch of OP50 is covered with cDCFDA. If required, use up to 150 &#181;L of cDCFDA for each plate. It is also imperative that the cDCFDA solution does not extend beyond the borders of the OP50 patch, since any cDCFDA that flows out of the OP50 patch will not be incorporated into the food, and will result in reduced staining intensity.</li>
		<li>Place the OP50 plates in a dark box on the bench top until all of the cDCFDA solution is absorbed into the bacterial patch (usually about 25 to 30 min).</li>
	</ol>
	</li>
	<li>Staining worms using cDCFDA
	<ol>
		<li>Once the cDCFDA has completely permeated the OP50 patch, place no more than 20 worms per plate and incubate the plates inverted at 20 &#176;C for a minimum of 14 h. 
		NOTE: It is recommended to place the cDCFDA plates in an opaque box to minimize exposure to light.</li>
		<li>To control for intake differences between experiments and to normalize cDCFDA signals, (recommended) co-stain with 3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron (2.5 &#181;L of a freshly made 1 mM solution), an acidotropic weak amine dye whose fluorescence is largely non-variant over the acidic pH spectrum (see Table of Materials for common names of reagents). 
		NOTE: Do not stain worms for less than 14 h since this may provide inadequate cDCFDA staining intensity and give a false interpretation of lysosomal pH.</li>
	</ol>
	</li>
	<li>Preparing slides for microscopy
	<ol>
		<li>Place two parallel strips of labeling tape about 3 inches apart on a smooth flat surface, such as a mirror (Figure 1).</li>
		<li>Coat the surface of the mirror with water repellent spray, and wipe surplus liquid off.</li>
		<li>Melt 2% agarose (dissolved in distilled H2O) in a microwave until completely liquefied, then place a 50 &#181;L drop of agarose between the two strips of tape and quickly cover the drop with a microscope slide, such that the slide is perpendicular to the strips of tape. After the agarose has solidified, forming a circular pad under the slide, flip the slide over and add 10 to 15 &#181;L of 5mM sodium azide (NaN3) onto the agarose pad. 
		NOTE: NaN3 is a cytochrome C inhibitor that when used in low concentrations, reversibly anesthetizes worms so that they will not move during imaging.</li>
		<li>Pick worms from the OP50 plate supplemented with cDCFDA, and transfer them to a clean NGM plate without any OP50. Allow the worms to move about for a few seconds to allow most of the OP50 to be removed from the surface of the worms, and then transfer the worms to the agarose pad containing 5 mM sodium azide.</li>
		<li>Immediately cover the agarose pad with a cover slip. Be sure to gently place the cover slip without applying too much pressure, since this can result in worms bursting. 
		NOTE: OP50 bacteria supplemented with cDCFDA fluoresce when visualized by microscopy, hence it is important to clean the worms as best as possible before placing them on the agarose pad containing sodium azide. If prepping more than 6 strains of C. elegans (including N2, wild type control), it is advisable to prepare the samples in batches of 6, so as to ensure that the worms do not dry out on the agarose pad. In some strains, the vulva starts to rupture after extended periods of time due to pressure from the cover slip, so it is important to plan accordingly.</li>
	</ol>
	</li>
	<li>Confocal microscopy 
	NOTE: Some microscopes have a built-in feature that automatically increases fluorescence intensity to compensate for variable levels of fluorescence. Such microscopes might not work well for imaging worms stained with cDCFDA, since they will inherently increase the fluorescence intensity of samples.
	<ol>
		<li>Perform imaging on a standard confocal microscope using excitation/emission wavelengths set to 488/530 nm for cDCFDA. Take single plane images (not Z-stacks) of intestinal lysosomes and use only lysosomes that are in focus (maximal signal intensity) for intensity quantification. 
		NOTE: Perhaps because of differences in dye availability, lysosomes of the intestinal cells directly posterior to the pharynx maintain cDCFDA staining intensity irrespective of genotype or age, hence care should be taken to avoid imaging lysosomes in these cells.</li>
		<li>Image the slide containing 2 day old wild type N2 worms first. While doing so, adjust the confocal imaging parameters such as laser power, pinhole, and aperture to ensure that the cDCFDA staining intensity is not oversaturated. 
		NOTE: Once these settings are set, do not change them for the entire duration of the imaging for that day.</li>
		<li>Capture 1024 x 1024 pixel images of a single plane of intestine (3 to 4 images per worm) using a 60X magnification lens. 
		NOTE: The cDCFDA emission spectrum in worms will yield a single prominent peak at 520 nm coinciding with its reported fluorescence spectrum20, providing a specific signal readout that is distinct from intestinal autofluorescence (Figure 3).</li>
		<li>Export raw confocal image (.lsm files) as .tiff files for each channel.</li>
		<li>Next, open ImageJ and click on File| Open to open the image file to be quantified. Once the image loads, use the oval shape selection tool in ImageJ to select a region of interest (ROI).</li>
		<li>Thereafter, click on Analyze| Measure to quantify fluorescence intensity for that ROI. Select 4&#8211;5 different in-focus lysosomes per image and calculate the average relative fluorescence intensity of each region of interest (ROI).</li>
		<li>For each image, measure the background fluorescence intensity in a region of the intestine between lysosomes. Subtract the background fluorescence intensity values from the lysosomal fluorescence intensity values.</li>
		<li>In total, collect around 30 to 50 individual normalized values for cDCFDA intensity (6 to 10 animals) for each strain being tested. Use these values to plot a box and whisker plot using any appropriate statistical software. 
		NOTE: Staining can vary considerably depending on factors like temperature, incubation time and the overall health/age of animals such that fluorescence intensity comparisons are only relevant within samples process in the same staining experiment. It is therefore important to process controls in every staining experiment. Calculate statistical differences (t-tests) using any appropriate statistical software.</li>
		<li>Image all strains from the same experiment (stained on the same day) in sequence, taking care not to change any of the imaging parameters.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Erjavec, N., Larsson, L., Grantham, J., Nystrom, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+aging+and+failure+to+segregate+damaged+proteins+in+Sir2+mutants.">Accelerated aging and failure to segregate damaged proteins in Sir2 mutants.</a> Genes Dev. 21 (19), 2410-2421 (2007).</li><li>Madeo, F., Eisenberg, T., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+for+the+avoidance+of+neurodegeneration.">Autophagy for the avoidance of neurodegeneration.</a> Genes Dev. 23 (19), 2253-2259 (2009).</li><li>Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+intracellular+protein-degradation+pathways+in+neurodegeneration.">The roles of intracellular protein-degradation pathways in neurodegeneration.</a> Nature. 443 (7113), 780-786 (2006).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313 (5793), 1604-1610 (2006).</li><li>Cuervo, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+aging:+the+importance+of+maintaining+&amp;#34;clean&amp;#34;+cells.">Autophagy and aging: the importance of maintaining &amp;#34;clean&amp;#34; cells.</a> Autophagy. 1 (3), 131-140 (2005).</li><li>Harrington, A. J., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+model+system+for+identifying+effectors+of+alpha-synuclein+misfolding+and+dopaminergic+cell+death+associated+with+Parkinson's+disease.">Caenorhabditis elegans as a model system for identifying effectors of alpha-synuclein misfolding and dopaminergic cell death associated with Parkinson's disease.</a> Methods. 53 (3), 220-225 (2011).</li><li>Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+amyloid+formation,+and+neurodegeneration:+a+critical+role+for+molecular+chaperones?.">Protein misfolding, amyloid formation, and neurodegeneration: a critical role for molecular chaperones?.</a> Neuron. 35 (1), 9-12 (2002).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+decline+in+chaperone-mediated+autophagy.">Age-related decline in chaperone-mediated autophagy.</a> J Biol Chem. 275 (40), 31505-31513 (2000).</li><li>Tonoki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+evidence+linking+age-dependent+attenuation+of+the+26S+proteasome+with+the+aging+process.">Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process.</a> Mol Cell Biol. 29 (4), 1095-1106 (2009).</li><li>Squier, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+protein+aggregation+during+biological+aging.">Oxidative stress and protein aggregation during biological aging.</a> Exp Gerontol. 36 (9), 1539-1550 (2001).</li><li>Sarkar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+enhance+autophagy+and+reduce+toxicity+in+Huntington's+disease+models.">Small molecules enhance autophagy and reduce toxicity in Huntington's disease models.</a> Nat Chem Biol. 3 (6), 331-338 (2007).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Boya, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+function+and+dysfunction:+mechanism+and+disease.">Lysosomal function and dysfunction: mechanism and disease.</a> Antioxid Redox Signal. 17 (5), 766-774 (2012).</li><li>Kim, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-aging+treatments+slow+propagation+of+synucleinopathy+by+restoring+lysosomal+function.">Anti-aging treatments slow propagation of synucleinopathy by restoring lysosomal function.</a> Autophagy. 12 (10), 1849-1863 (2016).</li><li>Vila, M., Bove, J., Dehay, B., Rodriguez-Muela, N., Boya, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+membrane+permeabilization+in+Parkinson+disease.">Lysosomal membrane permeabilization in Parkinson disease.</a> Autophagy. 7 (1), 98-100 (2011).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+intracellular+proteolytic+systems+change+with+age?.">How do intracellular proteolytic systems change with age?.</a> Front Biosci. 3, d25-d43 (1998).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+lysosomes+get+old.">When lysosomes get old.</a> Exp Gerontol. 35 (2), 119-131 (2000).</li><li>Gachet, Y., Codlin, S., Hyams, J. S., Mole, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=btn1,+the+Schizosaccharomyces+pombe+homologue+of+the+human+Batten+disease+gene+CLN3,+regulates+vacuole+homeostasis.">btn1, the Schizosaccharomyces pombe homologue of the human Batten disease gene CLN3, regulates vacuole homeostasis.</a> J Cell Sci. 118 (Pt 23), 5525-5536 (2005).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Preston, R. A., Murphy, R. F., Jones, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+vacuolar+pH+in+yeast+and+identification+of+acidification-defective+mutants.">Assay of vacuolar pH in yeast and identification of acidification-defective mutants.</a> Proc Natl Acad Sci U S A. 86 (18), 7027-7031 (1989).</li><li>Pringle, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy+methods+for+yeast.">Fluorescence microscopy methods for yeast.</a> Methods Cell Biol. 31, 357-435 (1989).</li><li>Baxi, K., Ghavidel, A., Waddell, B., Harkness, T. A., de Carvalho, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Lysosomal+Function+by+the+DAF-16+Forkhead+Transcription+Factor+Couples+Reproduction+to+Aging+in+Caenorhabditis+elegans.">Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis elegans.</a> Genetics. 207 (1), 83-101 (2017).</li><li>Colacurcio, D. J., Nixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+lysosomal+acidification-The+emerging+role+of+v-ATPase+in+aging+and+neurodegenerative+disease.">Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease.</a> Ageing Res Rev. 32, 75-88 (2016).</li><li>Kang, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+screening+identifies+ATM+as+a+target+for+alleviating+senescence.">Chemical screening identifies ATM as a target for alleviating senescence.</a> Nat Chem Biol. 13 (6), 616-623 (2017).</li><li>Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R., Finkbeiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+formation+reduces+levels+of+mutant+huntingtin+and+the+risk+of+neuronal+death.">Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.</a> Nature. 431 (7010), 805-810 (2004).</li><li>Brunk, U. T., Terman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial-lysosomal+axis+theory+of+aging:+accumulation+of+damaged+mitochondria+as+a+result+of+imperfect+autophagocytosis.">The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.</a> Eur J Biochem. 269 (8), 1996-2002 (2002).</li><li>Gerland, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autolysosomes+accumulate+during+in+vitro+CD8++T-lymphocyte+aging+and+may+participate+in+induced+death+sensitization+of+senescent+cells.">Autolysosomes accumulate during in vitro CD8+ T-lymphocyte aging and may participate in induced death sensitization of senescent cells.</a> Exp Gerontol. 39 (5), 789-800 (2004).</li><li>Poon, H. F., Vaishnav, R. A., Getchell, T. V., Getchell, M. L., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+analysis+of+differential+protein+expression+and+oxidative+modification+of+specific+proteins+in+the+brains+of+old+mice.">Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice.</a> Neurobiol Aging. 27 (7), 1010-1019 (2006).</li><li>Yin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+basis+of+lipofuscin,+ceroid,+and+age+pigment-like+fluorophores.">Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores.</a> Free Radic Biol Med. 21 (6), 871-888 (1996).</li><li>Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reduction+in+intestinal+cell+pHi+due+to+loss+of+the+Caenorhabditis+elegans+Na+/H++exchanger+NHX-2+increases+life+span.">A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.</a> J Biol Chem. 278 (45), 44657-44666 (2003).</li><li>Chakraborty, K., Leung, K., Krishnan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+lumenal+chloride+in+the+lysosome+is+critical+for+lysosome+function.">High lumenal chloride in the lysosome is critical for lysosome function.</a> Elife. 6, (2017).</li></ol>

The authors declare no conflict of interest.

A variety of cellular and molecular events contribute to aging, influenced by life history traits and genetic factors. Our recent study22 suggests that the reproductive cycle plays an important role in controlling the fitness of the soma through the regulation of lysosomal pH dynamics. We showed that lysosomal-mediated proteolysis is promoted while animals actively reproduce by upregulation of v-ATPase transcription, which in turn ensures acidic lysosomes. Upon the end of reproduction, v-ATPase expression drops, lysosomes alkalinize, and protein aggregates accumulate in these cells.
Disorders that impair lysosomal acidification have been shown to be caused by altered v-ATPase activity, and declining v-ATPase function in the brain has also been proposed to underlie various neurodegenerative diseases23. Our results further support the hypothesis that impaired v-ATPase function is among the causes of cellular senescence and aging24.
One cytological hallmark of aging cells is the progressive formation of misfolded and aggregated proteins in old cells1,25,26,27,28,29. As in post-reproductive C. elegans, similar protein aggregates are found in the lysosomal lumen of late passage mammalian cultured cells27,29. Since lysosomes are the terminal endpoint of the autophagic pathway, it is plausible that the continuous influx of damaged proteins and organelles into alkalinized lysosomes results in the functional collapse of these lysosomes, which then leads to other downstream events that ultimately contribute to cellular senescence.
The protocol described here has been designed to generate a simple and reproducible assessment of the relative acidity in intestinal lysosomes of live C. elegans. Because a narrow pH range (4.5&#8211;5) is required in these organelles for proper proteolysis, subtle increases in pH have dramatic consequences for cellular and tissue homeostasis17. The fluorescence characteristics and low pka of cDCFDA allows this dye to capture changes in acidity in this physiologically relevant pH window18. The concentration of cDCFDA recommended for staining lysosomes has been optimized to provide the best possible readout of changes in lysosomal pH based on our imaging parameters and imaging system. Even so, the results of cDCFDA staining might vary for different end users based on their imaging setup such that lysosomal pH changes might not be accurately reflected in the cDCFDA staining pattern. If this happens, the best course of action would be to use varying the concentrations of cDCFDA (10 to 100 mM) to determine the optimum concentration for imaging variations in wild type N2 worms at day 2 and day 8 post L4. The ideal cDCFDA concentration is one that stains lysosomes of young (day 2 post L4) worms efficiently without being oversaturated but also shows a decreased staining intensity for old (day 8 post L4) worms.
One of the limitations of the cDCFDA staining technique is its long incubation time. When worms are incubated at a particular time point, the results can be visualized only after ~24 h. It would be more efficient to develop a strain of C. elegans that provides a real-time readout of lysosomal pH, presumably using a pH-sensitive fluorophore tagged to a lysosomal membrane protein.
While there exist a few different techniques for assessing pH in the intestine of C. elegans, most of these techniques either assess the pH of the intestine as a whole30, or require injection into the body cavity, which is time-consuming and cumbersome31. cDCFDA can easily be applied for large scale genetic screens using feeding RNAi libraries to search for C. elegans mutants that show altered lysosomal pH or physiology. The only critical aspects of this method are the concentration of cDCFDA used and being sure to include a young (day 2 post L4) N2 control strain as a benchmark for configuring confocal imaging during every session.
To better understand the contribution of lysosomal pH to aging, it will be important to dissect the regulatory mechanisms behind the dynamic changes in lysosomal acidity in response to different cellular and environmental inputs. cDCFDA staining may be used as one strategy to extend this research in C. elegans.

The appearance of protein aggregates is widely accepted to be a hallmark of aging in eukaryotic cells1,2,3, and the formation of which is thought to be among the principle drivers of cellular senescence4,5,6,7. There is growing evidence that as cells age, protein catabolism is impaired, leading to an increase in protein aggregation. The collapse of proteolysis in aging cells involves an impairment of autophagy8 as well as proteasome-mediated protein degradation9. Finally, irreversible protein oxidation is increased in old cells, further impairing protein catabolism10.
Autophagy was initially thought to be a non-selective process for bulk degradation of damaged proteins, but recent studies have indicated that autophagy is highly selective to the catabolism of protein aggregates and dysfunctional organelles that are not amenable to degradation via other protein clearance mechanisms11. During the process of autophagy, damaged and aggregated proteins are sequestered into a double-membrane vesicle called the autophagosome. This autophagosome then fuses with the acidic organelles called lysosomes, which leads to degradation of the autophagosome cargo12. Lysosomes represent the end-point of the autophagic pathway and participate in different cellular processes such as membrane repair, transcriptional control and nutrient sensing; highlighting their centralized role in cellular homeostasis (reviewed in ref. 13). Several studies have shown an association between an age-dependent decrease in lysosomal function and various neurodegenerative disorders13. Consistently, restoring lysosomal function in older cells can delay the onset of aging-related phenotypes14,15. Studies of the composition of the intralumen milieu suggest that the collapse of lysosomal function in older cells is not due to a reduction in the production of lysosomal proteases16. Alternatively, it has been proposed that loss of intralysosomal acidity, a critical requirement of its enzymatic activity, may underlie the drop in lysosome-mediated proteolysis17. To be able to explore this hypothesis, it is essential to develop reagents and protocols to probe dynamic changes in lysosomal pH in live cells in a replicable and consistent manner.
The intestine of C. elegans is the major metabolic tissue in worms and it is a critical regulator of systemic homeostasis and lifespan. We have developed assays to evaluate changes in the acidity of the lumen of intestinal lysosomes of worms to determine how lysosome-mediated proteolysis contributes to aging. Though pH-sensitive fluorophores have been used previously in C. elegans to mark intestinal lysosomes, there hasn&#39;t yet been an effort to establish a successful protocol that can detect small increases in lysosomal pH in vivo18. Here, we provide a protocol that can be used to detect loss of lysosomal acidity in the intestinal cells of C. elegans using a simple and convenient feeding protocol that incorporates a pH-sensitive fluorophore (cDCFDA) into OP50 food.

<table><tbody><tr><td>OP50 (&lt;em&gt;E. coli&lt;/em&gt;)</td><td>Caenorhabditis Genetics Center</td><td></td><td>Order online at https://cgc.umn.edu/strain/OP50</td></tr><tr><td>5(6)-carboxy-2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein diacetate&amp;nbsp;</td><td>ThermoFisher</td><td>C369</td><td>Commonly known as cDCFDA</td></tr><tr><td>9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#039;H-2,2&#039;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td><td>ThermoFisher</td><td>L7528</td><td>Commonly known as Lysotracker Red</td></tr><tr><td>Confocal microscope (e.g. Zeiss LSM 510)</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>Download for free from https://imagej.nih.gov/ij/download.html</td></tr><tr><td>LB Broth powder</td><td>ThermoFisher</td><td>22700041</td><td></td></tr><tr><td>Bacto Agar</td><td>Sigma</td><td>A5306-1KG</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Bacto Peptone</td><td>Fisher Scientific</td><td>S71604</td><td></td></tr><tr><td>Cholesterol powder</td><td>Sigma</td><td>C3045&amp;nbsp;</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>449709</td><td></td></tr><tr><td>MgSO4</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>K3PO4</td><td>Sigma</td><td>P5629</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Cover Slips</td><td>ThermoFisher</td><td>3406</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A6013</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td></td></tr><tr><td>Mirror or other smooth flat surface</td><td></td><td></td><td></td></tr></tbody></table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

cDCFDA stains lysosomes in a pH-dependent manner, and its low pKa and ready uptake into lysosomes makes it an ideal pH sensor21. cDCFDA staining intensity is inversely proportional to lysosomal pH (i.e. staining intensity increases as pH decreases)18,22. cDCFDA signals are consistently weak in lysosomes of animals treated with 20 mM of chloroquine, an inhibitor of lysosomal acidification, and in worms depleted of v-ATPase, the protein complex on the membrane of lysosomes that is required for proton import22. These are important controls to use when assessing the relative levels of cDCFDA staining in other genetic backgrounds or treatments.
We also find no overt effects of cDCFDA exposure on the fitness or fertility of treated worms (data not shown). Using this protocol, we identified an endogenous loss of acidity in lysosomes of post-reproductive wild type worms. As shown in figure 3, the cDCFDA fluorescence signal is robust in the lysosomes of young (2 day post L4) reproducing C. elegans, signifying acidified lysosomes. The staining intensity reduces considerably upon alkalinization of lysosomes, as observed in old (8-day post L4) worms as well as in worms where the proton influx into these organelles is reduced (via knockdown of vha genes encoding v-ATPase subunits). Using this method, we detected a physiological loss of lysosomal acidity in the intestine of post-reproductive animals (diagram in Figure 2, fluorescence images in Figure 3). The reduced cDCFDA signal in intestines of day 8 (post-L4) worms pointed to alkalinized lysosomes and a possible impairment in protein clearance. This is in fact the case, as these animals significantly accumulate protein aggregates inside lysosomes at this life stage22. To validate a role of lumen de-acidification in this process, we co-stained young, reproducing animals that had been depleted by RNAi of two core components of the v-ATPase pump machinery (VHA-2 and VHA-8) and therefore should not properly import protons into lysosomes. As expected, vha-2 and vha-8 RNAi animals, even at the reproductive stage, showed reduced cDCFDA fluorescence signals comparable to those of post-reproductive wild type worms and consistent with alkalinized lysosomes (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57414/57414fig1.jpg" /> 
Figure 1: Preparing slides for microscopy. A simple step-by-step schematic showing the steps to be followed for preparing slides for microscopy.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57414/57414fig2.jpg" /> 
Figure 2: Use of pH-sensitive dyes to probe acidity in intestinal lysosomes of C. elegans. Diagram showing the patterns of intestinal cDCFDA fluorescence intensity in reproducing (day 2) and post-reproductive (day 8) worms. The top diagrams show whole worms. The boxes in the bottom represent zoomed in, projected diagrams of the midgut region, the intestinal lumen in the center. Lysosomes are depicted as vesicles of varying size in the cytoplasm of intestinal cells. Compare these schematic diagrams with Figure 3 images.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57414/57414fig3.jpg" /> 
Figure 3: cDCFDA staining images. Representative images showing day 2 and day 8 (post L4) C. elegans intestinal lysosomes (N2 wild type) stained with cDCFDA as well as intestinal lysosomes of vha-2 and vha-8 RNAi worms stained with cDCFDA (positive control). vha-2 and vha-8 encode subunits of the vacuolar ATPase (vATPase), the proton pump lining the lysosomal membrane that is responsible for acidification of the lysosomal lumen.

Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

assessing-lysosomal-alkalinization-intestine-live-caenorhabditis

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.9K Views.  University of Saskatchewan. A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (cDCFDA)

Video: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Biochemistry

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Neuroscience

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Biology

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Immunology and Infection

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Medicine

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

A Caenorhabditis elegans Model System for Amylopathy Study

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

Hand Dissection of Caenorhabditis elegans Intestines