We thank Dr. Jennifer Fox and the members of the Khalimonchuk laboratory for insightful comments. We acknowledge the support from the National Institutes of Health (R01 GM108975 to O.K. and T32 GM107001-01A1 to E.M.G.).

1. Preparation and collection of aged worms for cell isolation
NOTE: Explained below is the isolation of cholinergic neurons from the transgenic unc-17::GFP strain (OH13083) obtained from the Caenorhabditis Genetics Center (CGC) strain repository at the University of Minnesota. It is imperative to maintain sterile conditions to prevent contamination from fungi or bacteria.
<ol>
	<li>Prepare and synchronize worms via the bleaching method as described by T. Stiernagle16. For aging experiments, plate worms onto nematode growth medium (NGM) plates containing 25 &#181;M water solution of fluorodeoxyuridine (FuDR) to reduce egg production and arrest egg hatching. Inspect agar plates regularly to prevent contamination or egg hatching as this will cause unreliable results. 
	NOTE: For unc-17::GFP cells, typically three 100 mm x 15 mm NGM plates are used to isolate a sufficient amount of cells of interest16.</li>
	<li>Collect worms and prepare buffers for cell isolation.
	<ol>
		<li>Collect worms in a 15 mL conical tube. Wash worms from plate with 1.5 mL of M9 buffer (1 mM MgSO4, 85 mM NaCl, 42 mM Na2HPO4&#183;7H2O, 22 mM KH2PO, pH 7.0) and centrifuge for 5 min at 1,600 x g. Discard supernatant and wash worms with 1 mL of M9 buffer. Repeat centrifugation and wash for a total of five times to remove as much E. coli contamination as possible. 
		NOTE: Adding antibiotics (50 &#181;g/mL), such as ampicillin, at this step can help reduce bacterial contamination.</li>
		<li>For two samples, prepare 2 mL of sodium dodecyl sulfate-dithiothreitol (SDS-DTT) lysis buffer: 200 mM DTT, 0.25% (w/v) SDS, 20 mM HEPES (pH 8.0), and 3% (w/v) sucrose.</li>
		<li>Prepare 15 mL of isolation buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES [pH 7.3]) and store it on ice. 
		NOTE: Both the SDS-DTT lysis buffer and isolation buffer must be made fresh before each experiment.</li>
	</ol>
	</li>
	<li>Cuticle disruption and single cell isolation
	<ol>
		<li>Centrifuge animals collected in step 1.2.1 for 5 min at 1,600 x g. Remove all supernatant and suspend worms in 1 mL of M9 media and transfer to 1.5 mL microcentrifuge tube.</li>
		<li>Pellet worms with centrifugation at 1,600 x g for 5 min.</li>
		<li>Add 200 &#181;L of SDS-DTT lysis buffer to worms and incubate for 5 min at room temperature (RT). Worms should appear to be &#8220;winkled&#8221; along the body if viewed under a light microscope. 
		NOTE: Prolonged exposure to SDS-DTT lysis buffer may result in death of worms, which can be monitored by observing worms. Worms that are dead will elongate and will not curl.</li>
		<li>Add 800 &#181;L of ice-cold isolation buffer and mix by gently flicking tube.</li>
		<li>Pellet worms for 1 min at 13,000 x g at 4 &#176;C, remove supernatant and wash with 1 mL of isolation buffer.</li>
		<li>Repeat step 1.3.5 for a total of five times, carefully removing isolation buffer each time.</li>
		<li>Add 100 &#181;L of protease mixture from Streptomyces griseus (15 mg/mL) (Table of Materials) dissolved in isolation buffer to the pellet and incubate for 10&#8722;15 min at RT. 
		NOTE: As with the SDS-DTT lysis buffer, the extended protease digestion may result in excessive cleavage of proteins along the plasma membrane, preventing isolation of surface exposed GFP via magnetic beads.</li>
		<li>During incubation with protease mixture, apply mechanical disruption by pipetting samples up and down against the bottom of the 1.5 mL microcentrifuge tube with a 200 &#181;L micropipette tip for ~60&#8722;70 times. Keep the pipette tip against the wall of the microcentrifuge tube with constant pressure to properly disassociate cells.</li>
		<li>To determine the stage of digestion, remove a small volume (~1&#8722;5 &#181;L) of the digestion mixture, drop it on a glass slide and inspect it using a tissue culture microscope. After 5&#8722;7 min of incubation, worm fragments should have visibly reduced cuticle and a slurry of cells will be readily visible.</li>
		<li>Halt reaction with 900 &#956;L of commercially available cold Leibovitz&#8217;s L-15 medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (final concentration at 50 U/mL penicillin and 50 &#956;g/mL streptomycin).</li>
		<li>Pellet isolated fragments and cells by centrifugation for 5 min at 10,000 x g at 4 &#176;C. Wash the pelleted cells with 1 mL of cold L-15-supplemented media twice more to ensure that excess debris and cuticle is removed.</li>
		<li>Resuspend pelleted cells in 1 mL of L-15-supplemented media and leave on ice for 30 min. Take the top layer, approximately 700&#8722;800 &#181;L, to a microcentrifuge tube. This layer contains cells without cell debris and will be used in the subsequent isolation of cells of interest.</li>
		<li>Following manufacturer&#8217;s instructions, use an automated cell counter or hemocytometer to measure the cell density of 10&#8722;25 &#181;L of isolated cells.</li>
	</ol>
	</li>
</ol>
2. Isolation of GFP-positive cells via flow cytometry or anti-GFP magnetic beads
NOTE: There are two methods that can be deployed to isolate specific cell types. The first method is to use fluorescence-activated cell sorting (FACS), while the second method uses antibody-conjugated magnetic beads to pool cells expressing distinct plasma membrane-localized proteins. The latter method requires the localization of GFP to the plasma membrane, thus being available for interaction with the antibody-coupled magnetic bead.
<ol>
	<li>Isolation of GFP-positive cells by flow cytometry
	<ol>
		<li>From isolated cell suspension collected in step 1.3.12, centrifuge cells for 5 min at 10,000 x g at 4 &#176;C. Discard supernatant and suspend pelleted cells in L-15-supplemented medium to a cell density of no more than 6 x 106 cells/mL to avoid overloading the flow cytometer.</li>
		<li>Sort cells by GFP-positive expression using a flow cytometer capable of sorting samples. GFP-negative cells can be used as a control for non-cell type specific analysis. 
		NOTE: As mentioned above, an alternative approach to isolate GFP-positive cells may be used if the GFP-tagged protein of interest is localized to the cells&#8217; plasma membrane. In this case, the protocol described in section 2.2 can be employed.</li>
	</ol>
	</li>
	<li>Isolation of GFP-positive cells via anti-GFP magnetic beads
	<ol>
		<li>From isolated cell suspension collected in step 1.3.12, centrifuge cells for 5 min at 10,000 x g at 4 &#176;C. Discard supernatant and suspend cells in 485 &#181;L of L-15-supplemented medium. Add 15 &#181;L of ddH2O pre-washed &#945;-GFP antibody-coupled magnetic beads to the solution.</li>
		<li>Incubate cells with the magnetic bead slurry at 4 &#176;C for 1 h with very gentle turning. Excessive turning will damage the cells.</li>
		<li>After incubation, centrifuge sample for 5 min at 10,000 x g at 4 &#176;C, then wash pellet 2x with 1 mL of L-15-supplemented medium to remove GFP-negative cells.</li>
	</ol>
	</li>
	<li>Culturing of isolated cells
	<ol>
		<li>Plate isolated cells into a sterile 6-well multi-well plate. 
		NOTE: These plates may be coated with peanut lectin to increase cell attachment; however, if cells are to be used immediately, this step may be disregarded.</li>
		<li>Place the plate into a plastic container and incubate cells at 20 &#176;C with a damp wipe or soft tissue paper (add 50 &#181;g/mL ampicillin to ddH2O to ensure no bacterial growth on wipe) to add moisture to cells. Do not incubate in a CO2 incubator, as elevated CO2 levels may damage the cells.</li>
	</ol>
	</li>
</ol>
3. Fluorescent imaging of isolated GFP-positive cells
<ol>
	<li>Turn on the fluorescent bulb of an inverted microscope with fluorescence attachment and allow it to warm up for about 10 min.</li>
	<li>Remove cell culture from the incubator and set on stage of inverted microscope with florescence attachment. Slide the GFP filter into the grooves under the stage of the microscope.</li>
	<li>View the cells with a magnification of 50x. Successfully isolated GFP-positive cells will be readily visible. Take photos using the camera attachment on the microscope.</li>
</ol>
4. Extraction of RNA from isolated cells
NOTE: Extraction of RNA should be performed immediately after cell isolation to ensure high quality of material. Isolated RNA can be used to produce cDNA and subsequent measurement of transcript levels by quantitative PCR (qPCR). All tubes, tips, and reagents should be RNase-free, and workspace should be ethanol-washed prior to RNA extraction.
<ol>
	<li>From isolated cells collected at step 2.1.2, centrifuge cells in a 1.5 mL sterile, RNase-free microcentrifuge tubefor 5 min at 10,000 x g at 4 &#176;C. If cells are isolated via magnetic beads, follow cell collection as stated above.</li>
	<li>Using a micropipette, remove supernatant from centrifuged microcentrifuge tube and add 100 &#181;L of M9 medium to wash away L-15-supplemented medium. Centrifuge cells 5 min at 10,000 x g at 4 &#176;C and remove supernatant. To ensure all media are removed, repeat for a total of three washes carefully removing supernatant each time.</li>
	<li>Add 1 mL of phenol and guanidine isothiocyanate solution (Table of Materials) to cells and pipette the suspension up and down carefully. Incubate the mixture at RT for 5 min.</li>
	<li>Following incubation, add 250 phenol and guanidine isothiocyanate solution</li>
	<li>Centrifuge the solution for 5 min at 10,000 x g at 25 &#176;C.</li>
	<li>Carefully remove as much of the top aqueous layer with a micropipette as possible, without disturbing the bottom organic layers, and transfer the bottom layer to a new 1.5 mL RNase-free microcentrifuge tube. To the new tube, add 500 &#181;L of isopropanol and mix by inverting the tube. Incubate this solution at RT for 5 min.</li>
	<li>Centrifuge the tube at 14,000 x g for 20 min at 25 &#176;C.</li>
	<li>While keeping the samples on ice, remove as much isopropanol as possible with a micropipette, and gently add 1 mL of 70% (v/v) ethanol in RNase-free ddH2O to the tube. Be sure not to eject solution directly onto the bottom of the tube; apply ethanol/water mixture to the side of the tube.</li>
	<li>Centrifuge at 10,000 x g for 5 min at 25 &#176;C.</li>
	<li>Remove most of the ethanol and air dry on ice for 3&#8722;5 min. 
	NOTE: Ethanol should be removed after this step; however, careful inspection of bottom of the tube is important to ensure no ethanol is left.</li>
	<li>After the tube is air dried, add 15&#8722;20 &#181;L of RNase-free ddH2O and measure RNA concentration and purity using a spectrophotometer at 260 nm wavelength. The purity of the sample should be measured as a 260 nm/280 nm ratio. This ratio should be approximately &#62;2. 
	NOTE: Isolated RNA can be frozen and stored at -20 &#176;C. It is, however, highly preferential to use a cDNA synthesis kit as soon as possible to ensure high-quality cDNA production. Quantitative PCR can follow using instructions provided by the manufacturer of the thermocycler used in the laboratory.</li>
</ol>

<ol>
	<li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+cell+lineage+of+the+nematode+Caenorhabditis+elegans.">The embryonic cell lineage of the nematode Caenorhabditis elegans.</a> Developmental Biology. 100, 64-119 (1983).</li><li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+nervous+system+of+the+nematode+Caenorhabditis+elegans.">The structure of the nervous system of the nematode Caenorhabditis elegans.</a> Philosophical Transactions of the Royal Society B. 314, 1-340 (1986).</li><li>Zeng, H., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cell-type+classification:+challenges,+opportunities+and+the+path+forward.">Neuronal cell-type classification: challenges, opportunities and the path forward.</a> Nature Reviews in Neuroscience. 18, 530-546 (2017).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+protein+misfolding:+The+examples+of+Alzheimer&#8217;s+and+Parkinson&#8217;s+disease.">Cell biology of protein misfolding: The examples of Alzheimer&#8217;s and Parkinson&#8217;s disease.</a> Nature Cell Biology. 6, 1054-1061 (2004).</li><li>Choi, M. L., Gandhi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+of+protein+oligomerization+in+the+pathogenesis+of+Alzheimer&#8217;s+and+Parkinson&#8217;s+disease.">Crucial role of protein oligomerization in the pathogenesis of Alzheimer&#8217;s and Parkinson&#8217;s disease.</a> FEBS Journal. 285, 3631-3644 (2018).</li><li>Burman, J. L., 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=Mitochondrial+fission+facilitates+the+selective+mitophagy+of+protein+aggregates.">Mitochondrial fission facilitates the selective mitophagy of protein aggregates.</a> Journal of Cell Biology. 210, 3231-3247 (2017).</li><li>Shpilka, T., Haynes, C. M. <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+UPR:+mechanisms,+physiological+functions+and+implications.">The mitochondrial UPR: mechanisms, physiological functions and implications.</a> Nature Reviews Molecular Cell Biology. 19, 109-120 (2018).</li><li>Haynes, C. M., Petrova, K., Benedetti, C., Yang, Y., Ron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClpP+mediates+activation+of+a+mitochondrial+unfolded+protein+response+in+C.+elegans.">ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans.</a> Developmental Cell. 13, 467-480 (2007).</li><li>Walter, P., Ron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unfolded+protein+response:+from+stress+pathway+to+homeostatic+regulation.">The unfolded protein response: from stress pathway to homeostatic regulation.</a> Science. 334, 1081-1086 (2011).</li><li>Chen, C., Chen, Y., Jiang, H., Chen, C., Pan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron+aging:+learning+from+C.+elegans.">Neuron aging: learning from C. elegans.</a> Journal of Molecular Signal. 8 (14), 1-10 (2013).</li><li>Germany, E. 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=The+AAA-ATPase+Afg1+preserves+mitochondrial+fidelity+and+cellular+health+by+maintaining+mitochondrial+matrix+proteostasis.">The AAA-ATPase Afg1 preserves mitochondrial fidelity and cellular health by maintaining mitochondrial matrix proteostasis.</a> Journal of Cell Science. 131, jcs219956 (2018).</li><li>Zhang, S., Banerjee, D., Kuhn, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+larval+cells+from+C.+elegans.">Isolation and culture of larval cells from C. elegans.</a> PLOS One. 6 (4), e19505 (2011).</li><li>Morley, J. F., Morimoto, R. 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The authors have nothing to disclose.

The roundworm C. elegans is a well-established and powerful model to study neuronal health and disease2. With ample genetic tools to manipulate these animals and manageable quantity of precisely mapped various neuron types, a great deal of data can be collected with a relatively small quantity of material. Here, we outline an optimized method to isolate distinct neurons from whole animals. By disrupting the worm&#8217;s outer cuticle proteins, a slurry of various cell types can be isolate...

Over the past several decades, the metazoan model organism Caenorhabditis elegans has been a tremendous asset in the study of neurons, neuronal circuitry and its role in physiological and behavioral responses, and aging-associated neurodegenerative diseases. A unique feature of C. elegans is that the animals are transparent, allowing for the lineage of all adult somatic cells to be mapped1. C. elegans also harbors manageable quantity of neurons, leading to the morphology and connectivity of the nervous system being well understood2. The invariant cell lineage, short lifespan, and abundance of high-throughput genetic tools available for C. elegans make it an ideal model organism for the study of aging within different neuronal populations.
Aging of neurons and neurodegenerative disorders are complex, and each disorder has unique pathological signatures associated with it. However, for many of these disorders, such as Parkinson&#8217;s and Alzheimer&#8217;s disease, a common feature is the progressive load of misfolded proteins3,4,5. In both of these diseases, proteins misfold and aggregate within the cell to cause toxicity, ultimately leading to cell death4,5. For proper aging of neurons, the homeostasis of mitochondria, more specifically protein homeostasis, is important, as perturbations and dyshomeostasis can lead to neurodegeneration6,7,8. Cells are equipped with various mechanisms to maintain protein homeostasis, one being the unfolded protein response (UPR)&#8213;a classical pathway where signals from endoplasmic reticulum (ER) activate intracellular signal cascades, leading to a transcriptional response9. Akin to this UPRER, metazoans display a similar response to loss of mitochondrial protein homeostasis, the so-called mitochondrial UPR (UPRmt)7,8. C. elegans appears to be the most basal organism to have a confirmed and well-defined UPRmt pathway7.
The function/dysfunction of specific neuronal populations within a larger network can be difficult to assess due to their intrinsic connectivity and complexity3. However, there is often a need to study distinct neuronal populations due to cell type-specific diseases with complex pathologies, such as those associated with impaired cellular protein homeostasis. In C. elegans, specific neuronal populations can be genetically manipulated allowing for facile observation in vivo. The nervous system of C. elegans primarily consists of neurons, with a small percentage of glial cells. In adult hermaphrodite worms, there are approximately 302 neurons, subdivided into over 100 different classes2,10. Neurons such as cholinergic neurons predominate in neuromuscular junctions, while dopaminergic neurons are primarily involved in sensation10,11. As both motor activity and sensory abilities decline with age, it is important to detail mechanistic insights about defects in these individual neurons11,12. As such, there is a need for a simple and robust method to isolate intact cells of interest for subsequent ex vivo studies.
Here, we describe an optimized effective method for the rapid isolation of specific neuronal cells expressing green fluorescent protein (GFP) from C. elegans. This isolation method can be performed on larval, juvenile or adult worms. Isolation of cells from larval worms was previously published by Zhang et al.13 and will not be discussed here. An important note here is that all worms need to be at the same life stage to prevent over-digestion of the animal or contamination from animals in different life stages. Through both enzymatic and mechanical disruption of the nematode cuticle, an exoskeleton high in collagen and other structural proteins, a wide variety of cells can be isolated13,14. Cells can then be isolated via flow cytometry or antibody labeled magnetic beads. Typically, RNA is isolated via a phenol and guanidine isothiocyanate method to ensure the enrichment of desired cell population15. With much care these isolated cells can be maintained in a culture flask or multi-well dish. This method represents a unique and powerful tool in the study of specific neurons and has the capacity to isolate live and functional cells for further culturing.

<table>
	<tbody>
		<tr>
			<td>6-well plate</td>
			<td>Fisher Scientific</td>
			<td>12-556-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, Molecular Biology Grade</td>
			<td>VWR</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>496189</td>
			<td></td>
		</tr>
		<tr>
			<td>Contess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>USBiological</td>
			<td>D8070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Omega Scientific</td>
			<td>FB-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorodeoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>VWR</td>
			<td>BDH1133</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Amresco</td>
			<td>O395</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>USBiological</td>
			<td>P5110</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professionals</td>
			<td>7552</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Gibco</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>USBiological</td>
			<td>M2090</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>USBiological</td>
			<td>S5199</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>X190</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOCl (Bleach)</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Amresco</td>
			<td>O583</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomicen</td>
			<td>Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone Y</td>
			<td>USBiological</td>
			<td>P3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E</td>
			<td>Sigma</td>
			<td>7433</td>
			<td>protease mixture from Streptomyces griseus</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Amresco</td>
			<td>O227</td>
			<td></td>
		</tr>
		<tr>
			<td>SMT1-FLQC fluorescence stereomicroscope</td>
			<td>Tritech Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>USBiological</td>
			<td>S8010</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV One-Step synthesis kit</td>
			<td>ThermoFisher</td>
			<td>12594025</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>phenol and guanidine isothiocyanate solution</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Invitrogen</td>
			<td>T10Z82</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-GFP magnetic beads</td>
			<td>MBL</td>
			<td>D153-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of specific neuron populations from roundworm caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

The protocol described here allows for specific isolation of unc-17::GFP-positive cholinergic neurons from the roundworm C. elegans for subsequent ex vivo studies such as cell type-specific gene expression profiling and eventual short-term culturing for patch-clamp electrophysiology measurements.
Figure 1 shows unc-17::GFP-positive cholinergic neurons in their normal setti...

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Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

isolation-specific-neuron-populations-from-roundworm-caenorhabditis

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JoVE Journal

Developmental Biology

6.2K Views.  University of Nebraska. Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

Video: Isolation of Specific Neuron Populations from Roundworm 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 ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

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 ...

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.

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)

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

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.

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...