eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 threatened Litoria verreauxii alpina (for the Caspase 3/7 assay, Section 4), 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 them 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 calipers.</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 - 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, and 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 them to cool. Centrifuge the samples at 5,000 x g for 3 min, and then transfer the supernatant to individual 1.5 mL microcentrifuge tubes and store at -20 &#176;C until qPCR.</li>
		<li>Use quantitative PCR (qPCR) following Boyle et al. 2004 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 the 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 a 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 the manufacturer&#39;s instructions using a hemocytometer. 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 venter in individual 50 mL containers. Allow excess inoculum to collect into the base of the inoculation container. Leave each animal in an 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 solution, 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. 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 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 computer imaging software, by estimating the 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-dimensional 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>

<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>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 &amp;amp; Equipment</td><td>MW-113</td><td></td></tr><tr><td>Coomassie Bradford reagent</td><td>Pierce</td><td>23200</td><td></td></tr><tr><td>Caspase Glo 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>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&amp;prime;-CCTTGAT ATAATACAGTGTGCCATATGTC-3&amp;prime;)</td><td>Taqman</td><td></td><td>Individual design for primers and probe</td></tr><tr><td>Primer 5.8S Chytr (5&amp;prime;-TCGGTT CTCTAGGCAACAGTTT-3&amp;prime;)</td><td>Taqman</td><td></td><td>Individual design for primers and probe</td></tr><tr><td>Minor groove binder probe Chytr MGB2(5&amp;prime;-CGAGTCGAAC-3&amp;prime;)</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>

caspase-3/7 assay: a luminescence-based assay to quantify apoptosis by measuring caspase-3/7 activities in frog skin explants

Source: Brannelly, L. A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57345">Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis.</a> J. Vis. Exp. (2018).
This video describes an in vitro Caspase-3/7 luminescent assay to quantify cell death or apoptosis. The assay measures luminescence produced following caspase-3/7 cleavage of a pro-luminescent DEVD substrate. The luminescence generated is proportional to caspase-3/7 activity.

Caspase-3/7 Assay: A Luminescence-Based Assay to Quantify Apoptosis by Measuring Caspase-3/7 Activities in Frog Skin Explants

Source: Brannelly, L. A. et al., Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell ...

Caspase-3 and caspase-7 are proteases that recognize the DEVD tetrapeptide sequence in target proteins, cleaving them at the C-terminus of the aspartic acid residue. These play a prominent role in the apoptosis - programmed cell death - cascade.
To assess caspase-3/7 activity in apoptotic cells, begin with a tube containing a frog toe skin explant infected with apoptosis-inducing fungi. Add a suitable buffer to the tube and a few metallic beads.
Use bead-beating to disrupt the skin cells and release cellular contents, including caspase-3/7 proteins. Centrifuge the tube, pelleting cell debris, and organelles. Aspirate the protein-rich supernatant from the tube and pipette into the wells of a multiwell assay plate.
Supplement the wells with the luminogenic caspase reagent. This reagent consists of a proluminescent substrate containing the DEVD peptide in a buffered solution containing ATP, magnesium ions, and luciferase enzyme.
Mix the contents of the well and incubate. During incubation, caspase-3/7 cleaves the proluminescent substrate, releasing aminoluciferin - a substrate for luciferase - into the solution.
In the presence of ATP and magnesium ions, luciferase oxidizes the released aminoluciferin, generating a bioluminescent product. The amount of light produced during the reaction is proportional to the caspase activity.

caspase-37-assay-luminescence-based-assay-to-quantify-apoptosis

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

498 Views. Source: Brannelly, L. A. et al., Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis. J. Vis. Exp. (2018). This video describes an in vitro Caspase-3/7 luminescent assay to quantify cell death or apoptosis. The assay measures luminescence produced following caspase-3/7 cleavage of a pro-luminescent DEVD substrate. The luminescence generated is proportional to caspase-3/7 activity. 

Video: Caspase-3/7 Assay: A Luminescence-Based Assay to Quantify Apoptosis by Measuring Caspase-3/7 Activities in Frog Skin Explants - Concept

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

JoVE Journal

Neuroscience

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Caspase-3/7 Assay: A Luminescence-Based Assay to Quantify Apoptosis by Measuring Caspase-3/7 Activities in Frog Skin Explants

Transcript

Caspase-3/7 Assay: A Luminescence-Based Assay to Quantify Apoptosis by Measuring Caspase-3/7 Activities in Frog Skin Explants

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3