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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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 border="0" cellpadding="0" cellspacing="0" width="1166">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:475px;">OP50 (E. coli)</td>
			<td style="width:188px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td style="width:385px;">Order online at https://cgc.umn.edu/strain/OP50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">5(6)-carboxy-2&#8217;,7&#8217;-dichlorofluorescein diacetate&#160;</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">C369</td>
			<td>Commonly known as cDCFDA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:475px;">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</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">L7528</td>
			<td>Commonly known as Lysotracker Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Confocal microscope (e.g. Zeiss LSM 510)</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">ImageJ</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td>Download for free from https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">LB Broth powder</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">22700041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Agar</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">NaCl</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Peptone</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:119px;">S71604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cholesterol powder</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">C3045&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">CaCl2</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">449709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">MgSO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">K3PO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">P5629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Sodium Azide</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">DMSO</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:119px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cover Slips</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">3406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Agarose</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Incubator</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Mirror or other smooth flat surface</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></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

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...

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 ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.