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

Agradecemos às seguintes pessoas que ajudaram com criação e recolha de dados: Tegtmeier D. C. De Jong, J. Hawkes, Fossen K., S. Percival, M. McWilliams, L. Bertola, M. Stewart, s. Harney e T. Knavel; e M. Mercês para obter assistência com dissecações. Também gostaríamos de agradecer a M. McFadden, P. Harlow e Taronga Zoo para elevar o L. v. alpinae G. Marantelli para elevar o p. corroborre. Agradecemos a F. Pasmans, r. Martel para aconselhamento sobre ensaios de apoptose, C. Constantino, r. Kladnik e R. Webb para obter assistência com ensaio TUNEL, e T. Emeto e W. Weßels para ajudarem com o protocolo e kit de ensaio do caspase 3/7. Este manuscrito e o protocolo é adaptado de Brannelly et al 2017 Peer J22.

Universidade James Cook aprovado ética animal em aplicações A1875 para corroborre p. e A1897 e A2171 para L. v. alpina.
1. criação de animais e de monitoramento
<ol><li>Casa animais individualmente, em um ambiente adequado para a espécie, com uma água adequada, alimentação e horário de limpeza. Verifique os animais diariamente.<ol>
<li>Use indivíduos adultos do criticamente em perigo Pseudophryne corroboree (para o ensaio de TUNEL, seção 4) e o ameaça...

<ol>
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F., 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=Spread+of+chytridiomycosis+has+caused+the+rapid+global+decline+and+extinction+of+frogs.">Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs.</a> EcoHealth. 4, 125-134 (2007).</li><li>Voyles, J., 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=Pathogenesis+of+chytridiomycosis,+a+cause+of+catastrophic+amphibian+declines.">Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines.</a> Science. 326 (5952), 582-585 (2009).</li><li>Woodhams, D. 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R., 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=More+than+skin+deep:+Functional+genomic+basis+for+resistance+to+amphibian+chytridiomycosis.">More than skin deep: Functional genomic basis for resistance to amphibian chytridiomycosis.</a> Genome Biology and Evolution. 7 (1), 286-298 (2014).</li><li>Fites, J. 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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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Nós exploramos epidérmico apoptose e morte celular, como um mecanismo potencial de patologia de quitridiomicose a doença mortal ou um mecanismo de resistência a doenças em espécies sensíveis de Bd . Usamos dois métodos de avaliação de morte celular na epiderme, ensaio TUNEL para em situ análise de morte de células epidérmicas e ensaio do caspase 3/7 para monitoramento morte celular epidérmica em todo o progresso da infecção. Descobrimos que apoptose e morte celular é correlacionadas com...

Os anfíbios são actualmente uma as maiores perdas de biodiversidade global de qualquer vertebrado táxons1. Das principais causas destes declínios é a quitridiomicose de doença de pele fatal, causada por patógeno fungo Batrachochytrium dendrobatidis, Bd2. O patógeno infecta superficialmente a epiderme, que pode levar ao rompimento de função da pele, resultando em perda de eletrólito grave, parada cardíaca e morte3. Vários potenciais hospedeiro imune mecanismos contra Bd atualmente estão sendo estudados, tais como peptídeos antimicrobianos4,5, flora bacteriana cutânea6, imune celular receptores7,8, e linfócitos atividade9,10. No entanto, poucos estudos exploram se morte de apoptose e célula epidérmica é um mecanismo imune contra este agente patogénico mortal.
Morte celular, por meio de apoptose (morte celular programada) ou necrose (morte sem programado), a epiderme pode ser uma patologia de infecção de Bd . Pesquisas anteriores sugerem que Bd infecção pode induzir apoptose porque interrupção das junções intracelulares é observada quando a pele explantes estão expostos a zoospore sobrenadantes em vitro11. Além disso, alterações epidérmicas degenerativas em Bd-rãs infectadas são observadas usando microscopia eletrônica12,13. Transcriptomic análises indicam que as vias de apoptose são upregulated em pele infectada14, e anfíbios splenocytes sofrem apoptose quando eles são expostos a Bd sobrenadantes em vitro15. Apesar do volume crescente de evidências sugerindo que o Bd pode induzir apoptose e anfitrião pilha morte em vitro, estudos em vivo que explore ou quantificar a apoptose mecanismos através a progressão da infecção são escassos. Além disso, é desconhecido se o host usa apoptose como uma estratégia de defesa imune para combater infecção de Bd , ou se a apoptose é uma patologia da doença.
Neste estudo, tivemos como objetivo detectar morte celular epidérmica e apoptose em animais infectados na vivo usando dois métodos: ensaio de proteínas caspase 3/7 e terminal transferase-mediated dUTP nick rotulagem final (TUNEL) em situ do ensaio. Como cada ensaio detecta diferentes aspectos da célula morte16, juntos, esses métodos fornecem uma completa compreensão dos mecanismos envolvidos na morte celular e certifique-se de uma medida exata do efeito. O ensaio do caspase 3/7 quantifica a atividade das caspases efetoras 3 e 7, que permite a quantificação de ambas as vias de apoptose intrínseca e extrínseca. Em contraste, o ensaio TUNEL detecta fragmentação de DNA, que é causada por mecanismos de morte celular, incluindo apoptose, necrose e pyroptosis17. Nós usamos o ensaio do TUNEL para investigar o local da morte de pilha dentro da epiderme dos animais clinicamente infectados e não infectados usando três seções de pele diferentes: o dorso, o ventre e a coxa de Pseudophryne corroboree. Este método identifica o site anatômico de morte celular, bem como distinguir sua localização dentro de específicas camadas epidérmicas. Em seguida, usamos o ensaio do caspase 3/7 para conduzir uma quantificação de série de tempo da apoptose durante uma infecção de 8 semanas em Litoria verreauxii alpina. Tomamos amostras de ponta de dedo quinzenal dos mesmos animais e são capazes de correlacionar a carga de infecção do patógeno com atividade da caspase 3/7.

<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

Anfíbios estão enfrentando uma grande perda de biodiversidade global e uma das principais causas é a doença infecciosa quitridiomicose. Esta doença é causada por patógeno fungo Batrachochytrium dendrobatidis (Bd), que infecta e interrompe a epiderme de rã; no entanto, as alterações patológicas não foram explicitamente caracterizadas. Apoptose (morte celular programada) pode ser usado por patógenos para danificar o tecido do hospedeiro, mas também pode ser um mecanismo de anfitrião de resistência a doenças, para a remoção do agente patogénico. Neste estudo, podemos quantificar a morte de células epidérmicas de animais infectados e não infectados, usando dois diferentes ensaios: terminal transferase-mediated dUTP nick end-rotulagem (TUNEL) e caspase 3/7. Usando ventral, dorsal e o tecido da pele da coxa no ensaio de TUNEL, observamos pilha morte no células epidérmicas em situ de animais clinicamente infectados e comparar a morte celular com animais não infectados usando microscopia fluorescente. A fim de determinar como os níveis de apoptose na epiderme mudam no curso da infecção, nós removemos amostras de ponta de dedo do pé-quinzenalmente durante um período de 8 semanas e use um ensaio do caspase 3/7 com proteínas extraídas para quantificar a atividade dentro das amostras. Nós então correlacionar atividade caspase 3/7 com carga de infecção. O ensaio TUNEL é útil para a localização da célula morte em situ, mas é caro e tempo intensivo por amostra. O ensaio do caspase 3/7 é eficiente para tamanhos de amostra grande e tempo experiências do curso. No entanto, porque biópsias de ponta de dedo de rã são pequenas há extrato limitado disponível para padronização de amostra através de métodos de quantificação da proteína, tais como o ensaio de Bradford. Portanto, sugerimos que estimar a área de superfície da pele através da análise fotográfica de biópsias do dedo do pé para evitar consumir extratos durante a padronização da amostra.

Ensaio TUNEL
Havia mais células positivas do TUNEL nos animais infectados do que nos animais controle não infectados. A localização em situ do TUNEL positivo células diferiam em infectados e controlar animais. Nos animais controle, houve uma distribuição uniforme de TUNEL positivo células em toda a derme e epiderme da pele camadas em níveis baixos (ver figura 1A), mas nos ...

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

Podemos quantificar a morte celular epidérmica em sapos com quitridiomicose usando dois métodos. Primeiro, usar terminal transferase-mediated dUTP nick em situ histologia rotulagem final (TUNEL) para determinar diferenças entre animais clinicamente infectados e não infectados. Em segundo lugar, realizamos uma análise de série de tempo da apoptose infecção usando uma análise de proteínas caspase 3/7.

Usando o Terminal Transferase-mediada dUTP Nick End-rotulagem (TUNEL) e Caspase 3/7 ensaios a morte de células epidérmicas medida em sapos com quitridiomicose

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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