Name: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans (Video) | JoVE Journal
Uploaded: 2018-04-13T14:30:00.000+00:00
Duration: 6 min 18 s
Description: Learn to assess lysosomal alkalinization in Caenorhabditis elegans intestine using cDCFDA staining and confocal imaging. This simple, reproducible method helps researchers study autophagy and lysosomal function in vivo.

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5 minutes

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>

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</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 ex...

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

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 wo...

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)

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

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JoVE Journal

Developmental Biology

Source: Le, T. A. N., et al., Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans. J. Vis. Exp. (2019)This video demonstrates a fluorescence-based assay to quantify intestinal permeability in C. elegans exposed to Pseudomonas aeruginosa. Compared to controls fed Escherichia coli, pathogen-treated worms show increased fluorophore leakage into the body cavity, indicating epithelial barrier damage.

Assessment of Intestinal Permeability in C. elegans Following Bacterial Exposure

This video describes a colorimetric assay for lactate measurement in cell lysates by using the lactate dehydrogenase enzyme. This enzyme converts lactate into pyruvate, along with the conversion of NAD to NADH that reacts with tetrazolium dyes and gives a colored product that can be measured spectrophotometrically to quantitate the lactate.

Colorimetric Assay for Quantification of Lactate in Test Samples

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 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.

A Caenorhabditis elegans Model System for Amylopathy Study

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

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.

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 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 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

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.

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

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

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

Learn to assess lysosomal alkalinization in Caenorhabditis elegans intestine using cDCFDA staining and confocal imaging. This simple, reproducible method helps researchers study autophagy and lysosomal function in vivo.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

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