Name: using terminal transferase-mediated dutp nick end-labelling (tunel) and caspase 3/7 assays to measure epidermal cell death in frogs with chytridiomycosis
Uploaded: 2018-05-16T13:00:00
Description: Watch this Scientific Journal Video about Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis at JoV

Vi vil gerne takke følgende personer, der har bistået med dyrehold og dataindsamling: D. Tegtmeier, C. De Jong, J. Hawkes, K. Fossen, S. Percival, M. McWilliams, L. Bertoia, M. Stewart, N. Harney og T. Knavel; og M. Merces for bistand med dissektioner. Vi vil også gerne takke M. McFadden, P. Harlow og Taronga Zoo for at hæve L. v. alpinaog G. Marantelli til at hæve P. corroboree. Vi takker F. Pasmans, A. Martel for rådgivning om apoptose assays, C. Constantine, A. Kladnik og R. Webb for bistand med TUNEL assay, og T. Emeto og W. Weßels for hjælp med protokollen og kit til caspase 3/7 assay. Dette manuskript og protokollen er tilpasset fra Brannelly mfl 2017 Peer Jørgensen22.

James Cook University godkendt Dyreetik i programmer A1875 for P. corroboree og A1897 og A2171 for L. v. alpina.
1. husdyrhold og overvågning
<ol><li>Hus dyr individuelt, i et miljø, der er relevante for arterne, med en passende vand, fodring og rengøring tidsplan. Kontrollere dyrene dagligt.<ol>
<li>Bruge voksne individer af den kritisk truede Pseudophryne corroboree (for TUNEL Assay, afsnit 4) og truede Litoria verreauxii alpina (for Caspase 3/7 analy...

<ol>
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S., 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+invasive+chytrid+fungus+of+amphibians+paralyzes+lymphocyte+responses.">The invasive chytrid fungus of amphibians paralyzes lymphocyte responses.</a> Science. 342 (6156), 366-369 (2013).</li><li>Galluzzi, 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=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+cell+death+in+higher+eukaryotes.">Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.</a> Cell Death and Differentiation. 16 (8), 1093-1107 (2009).</li><li>Kelly, K. J., Sandoval, R. M., Dunn, K. W., Molitoris, B. A., Dagher, P. 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F., Berger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibians+with+infectious+disease+increase+their+reproductive+effort:+evidence+for+the+terminal+investment+hypothesis.">Amphibians with infectious disease increase their reproductive effort: evidence for the terminal investment hypothesis.</a> Open Biology. 6 (6), 1-24 (2016).</li><li>Webb, R., Mendez, D., Berger, L., Speare, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additional+disinfectants+effective+against+the+amphibian+chytrid+fungus+Batrachochytrium+dendrobatidis.">Additional disinfectants effective against the amphibian chytrid fungus Batrachochytrium dendrobatidis.</a> Diseases of Aquatic Organisms. 74 (1), 13-16 (2007).</li><li>Woods, A., Ellis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Histopathology: A Complete Reference. , (1994).</li><li>Brannelly, L. 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Vi udforskede epidermal apoptose og celledød som en potentiel mekanisme af patologi af dødbringende sygdom chytridiomycosis eller en mekanisme for sygdomsresistens i Bd modtagelige arter. Vi brugte to metoder til vurdering af celledød i epidermis, TUNEL assay for i situ epidermal celle død analyse og caspase 3/7 analysen til overvågning af epidermale celledød i hele forløbet af infektionen. Vi fandt, at celledød og apoptose er korreleret med infektion belastning og celledød er betydeligt højer...

Padder i øjeblikket oplever en de største tab af globale biodiversitet af ethvert hvirveldyr taxa1. En væsentlig årsag til disse afvisninger er fatal hud sygdom chytridiomycosis, forårsaget af de svampe patogen Batrachochytrium dendrobatidis, Bd2. Patogenet inficerer overfladen epidermis, som kan føre til afbrydelse af hudens funktion resulterer i alvorlige elektrolyt tab, hjertestop og død3. Forskellige potentielle vært immun mekanismer mod Bd er i øjeblikket ved at blive undersøgt som antimikrobielle peptider4,5, kutane bakterieflora6, immun celle receptorer7,8, og lymfocyt aktivitet9,10. Men få undersøgelser udforske om epidermal apoptose og celle død er en immun mekanisme mod denne dødbringende patogen.
Celledød, enten gennem apoptose (programmeret celledød) eller nekrose (unprogrammed død), i epidermis kan være en patologi af Bd infektion. Tidligere forskning tyder på, at Bd infektion kan inducere apoptose, fordi afbrydelse af intracellulære knudepunkter er observeret, når huden explants er udsat for zoospore analysere in vitro-11. Derudover degenerative epidermal ændringer i Bd-inficerede frøer er observeret ved hjælp af elektronmikroskopi12,13. Transkriptom analyser viser, at apoptose veje er upregulated i inficeret hud14, og padde splenocytes gennemgå apoptose, når de udsættes for Bd analysere in vitro-15. Trods den stigende mængde af beviser tyder på, at Bd kan inducere celle apoptose og vært død i vitro, i vivo studier, udforske eller kvantificere apoptose mekanismer gennem progression af infektion mangler. Yderligere, det er ukendt, hvis værten bruger apoptose som en defensiv immunsystemet strategi til bekæmpelse af Bd infektion, eller hvis apoptose er en patologi af sygdom.
I denne undersøgelse, vi har til formål at påvise epidermal celledød og apoptose i inficerede dyr i vivo ved hjælp af to metoder: caspase 3/7 protein assay og terminal-transferase-medieret dUTP nick ende-mærkning (TUNEL) i situ assay. Som hvert assay registrerer forskellige aspekter af celle død16, sammen disse metoder giver en fuld forståelse af de mekanismer, der er involveret i celledød, og sikre en nøjagtig måling af effekten. Caspase 3/7 assay kvantificerer aktiviteten af effektor caspases 3 og 7, som giver mulighed for kvantificering af både den indre og ydre apoptose veje. Derimod registrerer TUNEL assay DNA opsplitning, som er forårsaget af celle død mekanismer, herunder apoptose og nekrose pyroptosis17. Vi bruger TUNEL analysen for at undersøge placeringen af celledød i epidermis af både klinisk inficeret og ikke-inficerede dyr ved hjælp af tre forskellige hud sektioner: dorsum, venter og lår af Pseudophryne corroboree. Denne metode identificerer den anatomiske site af celledød, samt skelne dens placering inden for specifikke epidermale lag. Derefter bruger vi caspase 3/7 analysen til at gennemføre en gang serie kvantificering af apoptose i hele en 8-ugers infektion i Litoria verreauxii alpina. Vi tager tå tip prøver hver fjortende dag fra de samme dyr og er i stand til at korrelere patogen infektion belastning med caspase 3/7 aktivitet.

<table>
	<tbody>
		<tr>
			<td>POLARstar Omega</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>Luminescent plate reader</td>
		</tr>
		<tr>
			<td>384 well flat clear bottom plate</td>
			<td>Corning</td>
			<td>3707</td>
			<td></td>
		</tr>
		<tr>
			<td>384 well low flange white flat bottom plate</td>
			<td>Corning</td>
			<td>3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar Bacteriological (Oxoid)</td>
			<td>Fisher</td>
			<td>OXLP0011B</td>
			<td></td>
		</tr>
		<tr>
			<td>Formal-Fixx 10% Neutral Buffered Formalin</td>
			<td>Fisher</td>
			<td>6764254</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactose Broth (Oxoid)</td>
			<td>Fisher</td>
			<td>OXCM0137B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher</td>
			<td>BP328-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricane-S (MS-222)</td>
			<td>Fisher</td>
			<td>NC0872873</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>BP1421-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Invitrogen</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile rayon swab</td>
			<td>Medical Wire &#38; Equipment</td>
			<td>MW-113</td>
			<td></td>
		</tr>
		<tr>
			<td>ApopTag Red In Situ Apoptosis Detection Kit</td>
			<td>Merck Millipore</td>
			<td>S7165</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Bradford reagent</td>
			<td>Pierce</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase Glo&#160; 3/7</td>
			<td>Promega</td>
			<td>G8090</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma Aldrich</td>
			<td>H0887-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>1374248-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin hydrolysate Enzymatic</td>
			<td>Sigma-Aldrich</td>
			<td>G0262</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Saline), pH 7.2 (1X)</td>
			<td>Thermo/Life</td>
			<td>20-012-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepman</td>
			<td>Thermo/Life</td>
			<td>4318930</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Advanced Master Mix</td>
			<td>ThermoFisher</td>
			<td>4444556</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorox bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 Proof, Molecular Grade</td>
			<td>Fisher</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS Axio Scan florescent miscroscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Florescent microscope</td>
		</tr>
		<tr>
			<td>3.2mm stainless steel beads</td>
			<td>BioSpec</td>
			<td>11079132SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer ITSI-3 Chytr (5&#8242;-CCTTGATATAATACAGTGTGCCATATGTC-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Primer 5.8S Chytr (5&#8242;-TCGGTTCTCTAGGCAACAGTTT-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Minor groove binder probe Chytr MGB2(5&#8242;-CGAGTCGAAC-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Rotor-Gene qPCR Instruments</td>
			<td>Qiagen</td>
			<td></td>
			<td>qPCR machine</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5ml</td>
			<td>Fisher</td>
			<td>02-681-372</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture petri plates</td>
			<td>Nunc</td>
			<td>263991</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-beadBeater Zircornia-Silicate Beads, 0.5mm</td>
			<td>BioSpec</td>
			<td>11079105Z</td>
			<td></td>
		</tr>
	</tbody>
</table>

using terminal transferase-mediated dutp nick end-labelling (tunel) and caspase 3/7 assays to measure epidermal cell death in frogs with chytridiomycosis

Padder oplever et stort tab i biodiversiteten globalt og en af de vigtigste årsager er den smitsomme sygdom chytridiomycosis. Denne sygdom er forårsaget af de svampe patogen Batrachochytrium dendrobatidis (Bd), som inficerer og forstyrrer frøen epidermis; men patologiske ændringer er ikke blevet udtrykkeligt karakteriseret. Apoptose (programmeret celledød) kan bruges af patogener til at skade værten væv, men kan også være en vært mekanisme af sygdomsresistens for patogenet fjernelse. I denne undersøgelse, vi kvantificere epidermal celledød af smittede og ikke-inficerede dyr ved hjælp af to forskellige assays: terminal-transferase-medieret dUTP nick ende-mærkning (TUNEL) og caspase 3/7. Bruger ventrale, rygfinne, og lår hudvæv i TUNEL assay, vi observerer celle død i epidermal celler i situ af klinisk inficerede dyr og sammenligne celledød med ikke-inficerede dyr ved hjælp af fluorescerende mikroskopi. For at afgøre, hvordan apoptose niveauer i epidermis ændrede løbet af infektion vi fjerne tå-tip prøver hver fjortende dag over en 8-ugers periode, og bruge en caspase 3/7 assay med udvundne proteiner til at kvantificere aktivitet inden for prøverne. Vi derefter korrelere caspase 3/7 aktivitet med infektion belastning. TUNEL assay er nyttig for lokalisering af celle død i situ, men er dyre og tid intensive pr. prøve. Caspase 3/7 assay er effektiv til store stikprøvestørrelser og tid kursus eksperimenter. Men fordi frøen tå tip biopsier er små der er begrænset ekstrakt tilgængelige for prøven standardisering via protein kvantificering metoder, såsom Bradford assay. Vi foreslår derfor, estimering hud overflade område gennem fotografiske analyse af tå biopsier at undgå indtagelse af ekstrakter under prøven standardisering.

TUNEL Assay
Der var flere TUNEL positive celler i de angrebne dyr end i de ikke-inficerede kontroldyr. I situ -placeringen af TUNEL positive celler afveg i inficeret og kontrollere dyr. I kontroldyr var der en jævn fordeling af TUNEL positive celler i hele den dermal og epidermal hud lag på et lavt niveau (Se figur 1A), men i de angrebne dyr, TUNEL positive celler blev mere hyppi...

Watch this Scientific Journal Video about Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis at JoV

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Vi kvantificere epidermal celledød i frøer med chytridiomycosis ved hjælp af to metoder. Først bruger vi terminal-transferase-medieret dUTP nick ende-mærkning (TUNEL) i situ histologi for at bestemme forskellene mellem klinisk inficeret og ikke-inficerede dyr. Andet, foretager vi en tids-serie analyse af apoptose over infektion ved hjælp af en caspase 3/7 proteinanalyse.

Ved hjælp af Terminal-Transferase-medieret dUTP Nick ende-mærkning (TUNEL) og Caspase 3/7-undersøgelser til foranstaltning Epidermal celledød i frøer med Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...

The overall goal of this experiment is to observe the affect of amphibian fungal pathogen batrachochytrium dendrobatidis on apoptosis and cell death in the epidermal tissue of frogs. This method can help us understand wildlife disease such as if infection causes cell death or, conversely, if there are immune mechanisms against infection. The main advantage of this technique is that it can be performed on small tissue biopsy samples so that small individuals can be repeatedly sampled throughout the course of infection.To begin this procedure, prepare hydrophilic glass slides with sample tissue as outlined in the text protocol. To deparaffinize the tissue sections, wash the slides three times with xylene for five minutes each in a coplin jar. Next, wash the slides with 100%ethanol two times for five minutes each.After this, wash the slides with 95%ethanol and 70%ethanol for three minutes each. Then, wash the slides once with PBS for five minutes. To prepare experimental and negative control slides, pretreat the tissue by adding freshly diluted proteinase K directly to the slides.Incubate the slides for 10 minutes at room temperature. Then, wash the slides in PBS two times for two minutes each. To prepare positive control slides, pretreat the tissue with DN buffer then incubate the slides at room temperature for five minutes.After this, dissolve DNase one in DN buffer to a final concentration of 0.1 micrograms per milliliter. Add the prepared DNase solution to the slides. Incubate the slides for 15 minutes at room temperature.Then, wash the slides with distilled water in a coplin jar five times for three minutes each. Quench the reaction in 3%hydrogen peroxide in PBS for five minutes at room temperature. Next, wash the slides in PBS two times for two minutes each.Then, add equilibrium buffer to the tissue on the slides. Incubate for 10 seconds at room temperature. Then, tap off the excess liquid around the tissue section.Dilute terminal deoxynucleotidyl transferase enzyme in reaction buffer. Then, add it to the tissue on the experimental and positive control slides. Incubate in a humidified chamber for one hour at 37 degrees Celsius.After this, place the slides in a coplin jar containing the working concentration of stop buffer. Agitate for 15 seconds and then incubate the slides at room temperature for 10 minutes. Then, wash the slides with PBS three times for one minute each and tap off the excess liquid.Next, add the anti-digoxigenin conjugate, prewarmed to room temperature, directly onto the tissue. Incubate the slides for 30 minutes at room temperature in a humidified dark chamber. After the incubation is complete, wash the slides with PBS four times for two minutes each.Tap off any excess liquid. Then, add mounting medium to the slides. Add 15 microliters of DAPI solution to the mounting medium on the slides.Cover each slide with a cover slip and seal with nail polish. Allow the slides to dry in the dark. Using a fluorescent microscope, observe the slides immediately upon completion of the standing process.Take photos at 400 times at random intervals along each skin section. Count DAPI-stained blue cells to ensure at least 100 cells per skin section per animal are visible. Next, count the number of TUNEL-positive cells which appear as red single cells with clear cell edges.To begin, use an inverted light microscope at 40 times to take a photo of each of the frog's toes. Draw lines around the edge of the toe clip and use imaging software to estimate the area within the shape. To evaluate the surface area of the three-dimensional skin sample, multiply the estimated area by pi.To extract proteins from a frozen frog toe sample, place the sample in a 1.5 milliliter screw cap microcentrifuge tube containing 100 microliters of sample buffer and two stainless steel beads. Use a bead beater at maximum speed to lyse the cells. After this, place the tube on ice for three minutes.Then, centrifuge the tube to collect the supernatant. To perform the caspase 3/7 assay in a 384-well plate, add 10 microliters of sample buffer as blank and 10 microliters of protein extract in triplicate. Then, pipette 10 microliters of commercially available caspase 3/7 reagent to the protein extract and blank wells.Then, shake the plate gently for 15 seconds to mix the two reagents. Incubate at room temperature in dark for 30 minutes. Finally, use a luminescent plate reader to measure the luminescence of the sample.In this study, the infected animals depict significantly more TUNEL-positive cells in comparison to control animals as illustrated by red or pink-stained cells. In control animals, there are few TUNEL-positive cells, and they are not concentrated in the epidermis. In infected animals, the TUNEL-positive cells are more frequent in the epidermis.TUNEL-positive cells are more concentrated at and directly adjacent to the sights of infected cells. Also, there are more widespread TUNEL-positive cells over the epidermis and in epidermal layers that are deeper than where the fungal pathogen resided. In infected animals, there are more TUNEL-positive cells in all three skin types with a particularly high level in the thigh and venter skin.In the caspase assay, the infection intensity is positively correlated with caspase 3/7 activity. Additionally, caspase 3/7 activity in infected and uninfected animals differed over time. Caspase 3/7 activity decreased within the first few weeks after inoculation and then increased toward the end of week seven in infected animals.Caspase 3/7 activity remained constant in uninfected individuals. While attempting the procedure for the TUNEL assay, it's important to read the slides on the same day as the assay to avoid loss of sensitivity and to keep the slides in a cool dark place to protect the dyes. After watching this video, you should have a good understanding of how to detect apoptosis and cell death using two different detection methods in small tissue samples like amphibian skin.

Research

JoVE Journal

Immunology and Infection

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

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