eoe sub articles

EoE Sub Articles

1. Preparation and collection of aged worms for cell isolation
<ol>
	<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 KH2PO4, pH 7.0) and centrifuge for 5 min at 1,600 x&#160;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&#160;Escherichia coli&#160;contamination as possible. 
		&#8203;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 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (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 1.1.1 for 5 min at 1,600 x&#160;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&#160;g&#160;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 &#34;winkled&#34; 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&#160;g&#160;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&#160;Streptomyces griseus&#160;(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 green fluorescent protein (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&#39;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&#160;g&#160;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&#39;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 
<ol>
	<li>Isolation of GFP-positive cells by flow cytometry
	<ol>
		<li>From isolated cell suspension collected in step 1.2.12, centrifuge cells for 5 min at 10,000 x&#160;g&#160;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&#160;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.</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 double distilled water [ddH2O] to ensure no bacterial growth on wipe) to add moisture to cells. Do not incubate in a CO2&#160;incubator, as elevated CO2&#160;levels may damage the cells.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>FBS</td>
			<td>Omega Scientific</td>
			<td>FB-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</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>
	</tbody>
</table>

isolation of neurons from caenorhabditis elegans via fluorescence-activated cell sorting

Source: Germany, E. M., et al. <a href="https://app.jove.com/v/60145">Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans.</a> J. Vis. Exp. (2019)
This video demonstrates the isolation of fluorescently labeled neurons from Caenorhabditis elegans. A cell suspension is obtained from transgenic worms engineered to express fluorescent protein-fused vesicular transmembrane transporters in cholinergic neurons. The fluorescently labeled neurons are then isolated using fluorescence-activated cell sorting (FACS) and maintained in culture.

Isolation of Neurons from Caenorhabditis elegans via Fluorescence-Activated Cell Sorting

1. Preparation and collection of aged worms for cell isolation

	Collect worms and prepare buffers for cell isolation.
	
		Collect worms in a 15 mL ...

Take a suspension of cells obtained from transgenic Caenorhabditis elegans worms in a culture medium.
The suspension contains cells from various tissues, including neurons from the nervous system.
The transgenic worms were engineered to express a fluorescent protein conjugated to a vesicular transmembrane transporter. This transporter, present on synaptic vesicles carrying the neurotransmitter acetylcholine, is found in cholinergic neurons throughout the body.
Centrifuge the cell suspension and discard the supernatant. Resuspend the cells in a fresh culture medium for fluorescence-activated cell sorting, or FACS.
Analyze the cell suspension using FACS. The fluorescently labeled cholinergic neurons emit fluorescence upon exposure to light of an appropriate wavelength, separating them from other unlabeled cells.
Transfer the isolated neurons into a multi-well plate. Incubate the cells in a humidified chamber to maintain the moisture level.

isolation-neurons-from-caenorhabditis-elegans-via-fluorescence

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

62 ビュー. 出典:ドイツ、E.M.他回虫Caenorhabditis elegansからの特定のニューロン集団の分離。J. Vis. Exp.(2019年) このビデオは、線虫Caenorhabditis elegansからの蛍光標識ニューロンの分離を示しています。細胞懸濁液は、コリン作動性ニューロンで蛍光タンパク質融合小胞膜トランスポーターを発現するように操作されたトランスジェニック線虫から得られます。次に、蛍光標識されたニュー?...

ビデオ: 蛍光活性化細胞選別による線虫からのニューロンの単離 - 概念

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

C.エレガンスにおける大エキソファー小胞における生体内ニューロン集合体およびオルガネラ押出におけるスコアリングのための定量的アプローチ

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

JoVE Journal

神経科学

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

カエノラブディティス・エレガンスにおける樹状突起棘のイメージング

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

エレガンスにおける生理的ストレス応答の測定

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Isolation of Neurons from Caenorhabditis elegans via Fluorescence-Activated Cell Sorting

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Isolation of Neurons from Caenorhabditis elegans via Fluorescence-Activated Cell Sorting