Name: isolation of specific neuron populations from roundworm caenorhabditis elegans
Uploaded: 2019-08-06T15:00:00.000+00:00
Duration: 9 min 42 s
Description: Watch this Scientific Journal Video about Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans at JoVE.com

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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+longevity+in+Caenorhabditis+elegans+by+heat+shock+factor+and+molecular+chaperones.">Regulation of longevity in Caenorhabditis elegans by heat shock factor and molecular chaperones.</a> Molecular Biology of the Cell. 15, 657-664 (2004).</li><li>Pereira, 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=A+cellular+regulatory+map+of+the+cholinergic+nervous+system+of+C.+elegans.">A cellular regulatory map of the cholinergic nervous system of C. elegans.</a> eLife. 4, e12432 (2015).</li></ol>

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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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&#039;s L-15 Medium</td><td>Gibco</td><td>21083027</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>USBiological</td><td>M2090</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</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>&amp;alpha;-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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6.3K 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

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 ...

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 ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Neuroscience

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Biology

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

Hand Dissection of Caenorhabditis elegans Intestines

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

Hand Dissection of Caenorhabditis elegans Intestines

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

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

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells

Source: Germany, E. M., et al. <a href="https://www.jove.com/video/60145/isolation-specific-neuron-populations-from-roundworm-caenorhabditis">Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans</a>. J. Vis. Exp. (2019).
This video describes a method to dissociate whole worms into a single-cell suspension suitable for FACS or immunoprecipitation of intact cells.

Dysocjacja pojedynczych komórek Caenorhabditis elegans: metoda izolowania żywych komórek

Source: Germany, E. M., et al. Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans. J. Vis. Exp. (2019).
This video describes a ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

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Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

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

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

A Method for Culturing Embryonic C. elegans Cells

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Hand Dissection of Caenorhabditis elegans Intestines

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Dysocjacja pojedynczych komórek Caenorhabditis elegans: metoda izolowania żywych komórek