We thank the following people who assisted with husbandry and data collection: D. Tegtmeier, C. De Jong, J. Hawkes, K. Fossen, S. Percival, M. McWilliams, L. Bertola, M. Stewart, N. Harney, and T. Knavel; and M. Merces for assistance with dissections. We would also like to thank M. McFadden, P. Harlow and Taronga Zoo for raising the L. v. alpina, and G. Marantelli for raising the P. corroboree. We thank F. Pasmans, A. Martel for advice on apoptosis assays, C. Constantine, A. Kladnik and R. Webb for assistance with TUNEL assay, and T. Emeto and W. We&#223;els for help with protocol and kit for caspase 3/7 assay. This manuscript and protocol is adapted from Brannelly et al 2017 Peer J22.

James Cook University approved animal ethics in applications A1875 for P. corroboree and A1897 and A2171 for L. v. alpina.
1. Animal Husbandry and Monitoring
<ol>
	<li>House animals individually, in an environment appropriate for the species, with an appropriate water, feeding and cleaning schedule. Check animals daily.
	<ol>
		<li>Use adult individuals of the critically endangered Pseudophryne corroboree (for the TUNEL Assay, Section 4) and the threatened Litoria verreauxii alpina (for the Caspase 3/7 assay, Section 5), donated from captive breeding facilities. Maintain the animals at 15 - 18 &#176;C, on a moss and gravel substrate, mist them daily with reverse osmosis water, and feed 3 times weekly with gut loaded crickets.</li>
	</ol>
	</li>
	<li>Collect data on each individual once weekly. Swab the individuals for Bd as described in Step 2.1. Weigh each animal to the neared 0.1 g using a scale, and measure their snout to vent (SVL) length to the nearest 0.01 mm using dial callipers.</li>
	<li>After inoculation (as described in Section 3), check animals daily for clinical signs of infection (irregular skin slough, inappetence, leg redness, lethargy, or delayed righting reflex) in compliance with animal ethics guidelines. Euthanize animals if clinical signs and morbidity are present.
	<ol>
		<li>Euthanize with an overdose of tricaine methanesulfonate (MS222) (0.1% w/v MS222 to pH 6 -&#160;7 with sodium bicarbonate in aged tap water). Keep animals in MS222 for 10 min after all motion ceases and they display no response to stimuli.</li>
	</ol>
	</li>
</ol>
2. Testing for Bd Infection
<ol>
	<li>Swabbing for Bd
	<ol>
		<li>Swab each animal with a new sterile rayon swab (one swab per animal). Use the following swabbing method: five times on the venter, five times on each thigh, side, and limb. This is a total of 45 strokes.</li>
		<li>Gently rotate the swab during and between strokes to ensure effective capture of DNA. Break the swab tip off into 1.5 mL microcentrifuge tubes and store at -20 &#176;C until extraction.</li>
	</ol>
	</li>
	<li>DNA Extraction and qPCR assay
	<ol>
		<li>Extract genomic DNA from the swabs by adding 50 &#181;L of a commercially available DNA extraction reagent with 30 - 40 mg of 0.5 mm silica beads. Bead beat the samples in a bead beater cell disrupter at maximum speed for 2 min. Following the bead beater step, centrifuge the samples at 2,000 x g for 1 min.</li>
		<li>Incubate the samples at 100 &#176;C for 10 min, and allow to cool. Centrifuge the samples at 5,000 x g for 3 min, and then transfer the supernatant to individual 1.5 mL micro centrifuge tubes and store at -20 &#176;C until qPCR.</li>
		<li>Use quantitative PCR (qPCR) following Boyle et al. 200418 to analyze the infection load. Use the following modifications:
		<ol>
			<li>Dilute DNA extraction samples 6:100 with double deionized water. Add 0.7 &#181;L of bovine serum albumin (BSA) to each well to help prevent PCR inhibition. Run each sample in singlicate. On each plate use a negative (no template) control and a positive control with a series of dilution standards to estimate zoospore (infection) load.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Inoculation
<ol>
	<li>Culture Bd
	<ol>
		<li>Prepare culture broth by adding 16 g of tryptone, 2 g of gelatin hydrolysate, and 4 g of lactose (TGHL) to 1 L of deionized water. Autoclave (121 &#176;C for 40 min) and allow broth to cool.</li>
		<li>Inoculate Bd isolate (in this case, isolated from New South Wales isolate, AbercrombieR-L.booroologensis-2009-LB1, Passage number 11) in a TGHL broth and grow for 7 d at 23 &#176;C, then transfer the broth culture to TGHL agar plates.</li>
		<li>To make plates, add 16 g of tryptone, 2 g of gelatin hydrolysate, 4 g of lactose (TGHL), and 10 g of bacteriological agar to 1 L of deionized water and autoclave (121 &#176;C for 40 min). Once the mixture is cool enough to touch, but before it solidifies, pour the TGHL agar into 92 mm diameter culture plates so they are 1/4 to 1/3 full, in a Class II biological safety cabinet.</li>
		<li>When the agar has solidified and cooled completely, inoculate each plate with 0.5 mL of Bd from the liquid broth, and spread evenly. Allow Bd broth mixture to dry on plate for approximately 1 h and then seal plates with plastic paraffin film. Incubate plates agar side down at 23 &#176;C for 5 - 7 d.</li>
	</ol>
	</li>
	<li>Flood plates for zoospore inoculation solution
	<ol>
		<li>Check zoospore motility under an inverted light microscope to ensure viability daily before inoculation. When zoospore release is high, with many zoospores swimming outside the sporangia, the culture is ready for inoculation.</li>
		<li>Flood each plate with 3 mL of aged tap water or artificial pond water, by pouring the water into the plate and incubating at room temperature for 10 min to allow the zoospores to release into the water. After the incubation period, decant the zoospore suspension into a new sterile container.</li>
		<li>Estimate zoospore concentration following manufacturer&#39;s instructions using a haemocytometer. Once the concentration of the zoospore suspension is known, dilute the suspension to a concentration of 1 x 106 zoospores per 3 mL with aged tap water.</li>
	</ol>
	</li>
	<li>Inoculate animals
	<ol>
		<li>Inoculate each animal by pouring 3 mL of inoculum mixture over its venter19 in individual 50 mL containers. Allow excess inoculum to collect into the base of the inoculation container. Leave each animal in individual inoculation container for 24 h to ensure infection, and after the 24 h inoculation, return each individual to a disinfected terrarium.
		<ol>
			<li>Disinfect terraria using a 13% volume/volume (v/v) commercial bleach solution20, rinse at least twice with water, then allow to dry for no less than 24 h.</li>
		</ol>
		</li>
		<li>For Bd-negative controls, mock inoculate the animals using the same methods as in 3.2 - 3.3, but flood Bd-free agar plates with 5 mL of aged tap water instead of Bd inoculated agar plates.</li>
	</ol>
	</li>
</ol>
4. TUNEL Assay
<ol>
	<li>Euthanize animals that are showing clinical signs of chytridiomycosis, as described in step 1.3.1, and an equal number of Bd-negative control animals.</li>
	<li>Dissect skin (dorsal, ventral, and thigh) samples from each animal. Fix the skin samples for 2 h in 4% v/v phosphate buffered formaldehyde. The short and consistent fixing time allows the tissues to be fully fixed, but also allows for effective and accurate immunohistochemistry staining. Then transfer to 80% ethanol until embedding for sectioning.</li>
	<li>Embed skin in paraffin wax for histological preparation following standard methods21. Briefly, the protocol is as follows:
	<ol>
		<li>Dehydrate tissues in a graded series of ethanol, and clear the ethanol with xylene. Embed the tissue in paraffin wax, and place all three skin samples for each individual in one paraffin block.</li>
	</ol>
	</li>
	<li>Section skin using a microtome following standard histological preparation21. Section the tissue serially at 5 &#181;m and then affix to hydrophilic glass slides. Place four serial histosections per skin type per slide. Make three slides per individual with serial skin sections, remember that each slide has four sections of each tissue affixed.</li>
	<li>Stain the slides in the following order:
	<ol>
		<li>Stain the first slide with hematoxylin followed by eosin counterstaining (H&#38;E). This slide is to visualize the location of Bd sporangia within the skin.</li>
		<li>Stain the second slide following a commercially available TUNEL assay for histological preparation and follow manufacturer&#39;s instructions, which are described below.
		<ol>
			<li>First, deparaffinize the tissue sections in a coplin jar by washing the slides with three changes of xylene for 5 min per wash, and follow with two changes of 100% ethanol for 5 min each wash. Follow with one wash of 95% ethanol for 3 min and then 70% ethanol for 3 min. Finish with one wash of PBS for 5 min.</li>
			<li>Pretreat the tissue with freshly diluted protein digesting enzyme (proteinase K at a concentration of 20 &#956;g/mL diluted in PBS), and add directly to the slide. Allow to incubate for 15 min. Wash with two changes of PBS in a coplin jar for 2 min each.</li>
			<li>Tap off the excess liquid and quench in 3.0% hydrogen peroxide in PBS in a coplin jar for 2 min at room temp. Rinse twice with PBS, for five minutes each wash.</li>
			<li>Tap off the excess liquid, then apply 75 &#181;L/5 cm2 of the equilibrium buffer directly onto the tissue on the slide. Incubate for 10 s. Tap off the excess liquid around the section and apply 55 &#181;L/5 cm2&#160;of working terminal deoxynucleotidyl tranferase (TdT) enzyme. Incubate in a humidified chamber for 1 h at 37 &#176;C.</li>
			<li>After the TdT incubation, put the slide in a coplin jar of working strength stop/wash buffer, agitate for 15 s and incubate for 10 min at room temperature. Wash the slide with three changes of PBS for 1 min per wash. Tap off the excess liquid.</li>
			<li>Apply anti-digoxigenin conjugate (rhodamine) that has been warmed to room temperature to the tissue, 65 &#181;L/5 cm2. Incubate in a humidified chamber for 30 min at room temp and avoid exposure to light. Wash with four changes of PBS for 2 min per wash. Tap off the excess liquid.</li>
			<li>Finish by adding 15 &#181;L of 0.5 - 1 &#181;g/mL DAPI (4&#39;,6-diamidino-2-phenylindole) in slide mounting medium to the slide, which acts as a counter stain. Then cover with a cover slip and seal with nail polish or rubber cement. Allow slides to dry in the dark as the assay is light sensitive.</li>
		</ol>
		</li>
		<li>Stain the third slide is stained using the TUNEL assay and DAPI counterstain, but use as a positive and negative control for each sample. 
		NOTE: Of the 4 histosections on the control slides, the first 2 are positive control replicates and the last 2 are negative control replicates.
		<ol>
			<li>For the positive controls, instead of step 4.5.2.2, pre-treat the tissue with DN buffer (30mM trizma base, pH 7.2, 4mM MgCl2, 0.1mM DDT) and let incubate at room temperature for 5 minutes. Then dissolve Dnase I in DN buffer for a final concentration of 0.1ug/mL, and apply it directly to the slide. Incubate for 15 minutes at room temperature.&#160; Then, wash the slide with 5 wash of dH2O for 3 minutes each wash. Blot off the excess liquid. Then resume TUNEL assay as directed in section 4.5.2.3.</li>
			<li>For the negative controls, do not add the terminal deoxynucleotidyl tranferase (TdT) enzyme to those samples (as described in section 4.5.2.4).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Observe TUNEL slides immediately upon completion of the assay.
	<ol>
		<li>Take photos at random intervals along each skin section at 200X using a fluorescent microscope, using filters for both the rhodamine stain, which reveals the apoptotic cells and appears red, and DAPI which reveals all nuclei and appears blue, so that an overlay of cells can be generated. Ensure at least 100 cells are photographed per skin section per sample. Store the slides in a dark microscope box at -20 &#176;C.</li>
		<li>Count cells (blue DAPI stained) per image. Ensure at least 100 cells are counted per skin section. To reach 100 cells, count all cells within the image. If less then 100 cells are present in one image, count another image until at least 100 cells are reached per skin section per animal.</li>
		<li>Next, count the number of TUNEL positive cells in the same images (red rhodamine stained). TUNEL positive cells, which indicate apoptosis and not necrosis or background, are single cells with clear cell edges (membranes are intact with no lysis). Sometimes the cells shrink to form apoptotic bodies.</li>
		<li>Locate the areas counted in the TUNEL assay and analyze the corresponding regions on the H&#38;E stained histosections. Confirm that the H&#38;E stained sections correspond to sites of Bd infection, which ensures Bd-infected sites are used when counting apoptotic cells in the TUNEL assay.</li>
	</ol>
	</li>
</ol>
5. Caspase 3/7 Assay
<ol>
	<li>Collect toe tips from each animal (both Bd- exposed and Bd- negative) once weekly through week 3 post inoculation, and fortnightly through the end of the experiment, up to 8 toes per individual.
	<ol>
		<li>Cut the toe tip at the second phalange with flame-sterilized scissors, and place in a 1.5 mL microtube and freeze immediately at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Extract proteins from frozen toe sample.
	<ol>
		<li>Place samples in 100 &#181;L of sample buffer (25 mM HEPES pH 7, 5 mM MgCl2) with two stainless steel beads (3.2 mm) in a 1.5 mL screw cap microtube. Lyse samples by 4 cycles of 1 min bead beating at maximum speed (in the same bead beater as used in step 1.2.1) followed by 3 min on ice. After lysis, centrifuge samples at 12,000 x g and 4 &#176;C for 5 min. Collect supernatant to use in the assay.</li>
	</ol>
	</li>
	<li>Quantify protein concentration per sample using the Bradford assay.
	<ol>
		<li>In a 384 well plate, mix 10 &#181;L of protein extract with 10 &#181;L of Bradford reagent, and incubate for 2 min at room temperature. Perform each sample in duplicate, along with a series of 5 fold BSA dilution standards. Read the absorbance at 595 nm on an absorbance plate reader.</li>
	</ol>
	</li>
	<li>Alternatively, if the toe tip samples are too small (the protein concentration is near the lowest standard as determined from the Bradford assay in step 4.3.1, or if the frogs that are sampled are under 3 g total weight), standardize samples by estimating skin surface area using photographs, instead of the Bradford assay.
	<ol>
		<li>Before extracting the proteins from the sample for the caspase assay, photograph each toe at 40X magnification using an inverted light microscope.</li>
		<li>Analyze the toe sample using a computer imaging software, by estimating area of the toe. Do this by drawing a line around the edge of the toe clip, and have the imaging software estimate area within the shape. Then multiply that area by pi (3.14), which will approximate the surface area of the 3-dimentional skin sample (as length x cross-sectional area (pi x diameter) gives the outer surface area of a tube).</li>
	</ol>
	</li>
	<li>Perform the caspase 3/7 assay.
	<ol>
		<li>In a 384 well luminescence plate, add 10 &#181;L of commercially available caspase 3/7 reagent and 10 &#181;L of protein extract. Run each sample in triplicate.</li>
		<li>Mix the reagents by shaking the plate slowly for 15 s. Incubate the plate away from light for 30 min at room temperature. Measure luminescence using a luminescent plate reader.</li>
	</ol>
	</li>
</ol>

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The authors declare no competing financial interests.

We explored epidermal apoptosis and cell death as a potential mechanism of pathology of the deadly disease chytridiomycosis or a mechanism of disease resistance in Bd susceptible species. We used two methods of assessing cell death in the epidermis, TUNEL assay for in situ epidermal cell death analysis, and caspase 3/7 assay for monitoring epidermal cell death throughout the progress of infection. We found that cell death and apoptosis are correlated with infection load and cell death is significantly h...

Amphibians are currently experiencing one the greatest losses of global biodiversity of any vertebrate taxa1. A major cause of these declines is the fatal skin disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis, Bd2. The pathogen superficially infects the epidermis, which can lead to the disruption of skin function resulting in severe electrolyte loss, cardiac arrest, and death3. Various potential host immune mechanisms against Bd are currently being studied, such as antimicrobial peptides4,5, cutaneous bacterial flora6, immune cell receptors7,8, and lymphocyte activity9,10. However, few studies explore whether epidermal apoptosis and cell death is an immune mechanism against this deadly pathogen.
Cell death, either through apoptosis (programmed cell death) or necrosis (unprogrammed death), in the epidermis may be a pathology of Bd infection. Previous research suggests that Bd infection may induce apoptosis because disruption of intracellular junctions is observed when skin explants are exposed to zoospore supernatants in vitro11. Additionally, degenerative epidermal changes in Bd-infected frogs are observed using electron microscopy12,13. Transcriptomic analyses indicate that apoptosis pathways are upregulated in infected skin14, and amphibian splenocytes undergo apoptosis when they are exposed to Bd supernatants in vitro15. Despite the growing volume of evidence suggesting that Bd can induce apoptosis and host cell death in vitro, in vivo studies that explore or quantify apoptosis mechanisms through the progression of infection are lacking. Further, it is unknown if the host uses apoptosis as a defensive immune strategy to combat Bd infection, or if apoptosis is a pathology of disease.
In this study, we aimed to detect epidermal cell death and apoptosis in infected animals in vivo using two methods: caspase 3/7 protein assay, and terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ assay. As each assay detects different aspects of cell death16, together these methods provide a full understanding of the mechanisms involved in cell death, and ensure an accurate measure of the effect. The caspase 3/7 assay quantifies the activity of effector caspases 3 and 7, which enables quantification of both the intrinsic and extrinsic apoptosis pathways. In contrast, the TUNEL assay detects DNA fragmentation, which is caused by cell death mechanisms including apoptosis, necrosis and pyroptosis17. We use the TUNEL assay to investigate the location of cell death within the epidermis of both clinically infected and uninfected animals using three different skin sections: the dorsum, the venter and the thigh of Pseudophryne corroboree. This method identifies the anatomical site of cell death, as well as distinguishing its location within specific epidermal layers. We then use the caspase 3/7 assay to conduct a time series quantification of apoptosis throughout an 8-week infection in Litoria verreauxii alpina. We take toe tip samples fortnightly from the same animals and are able to correlate pathogen infection load with caspase 3/7 activity.

<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>
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			<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>
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		<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

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

TUNEL Assay
There were more TUNEL positive cells in the infected animals than in the uninfected control animals. The in situ location of TUNEL positive cells differed in infected and control animals. In control animals, there was an even distribution of TUNEL positive cells throughout the dermal and epidermal skin layers at low levels (See Figure 1A), but in the infected animals, t...

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

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

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

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

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Research

JoVE Journal

Immunology and Infection

10.4K Views.  James Cook University. We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

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

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...