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

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

1. House animals individually, in an environment appropriate for the species, with an appropriate water, feeding, and cleaning schedule. Check animals daily.
   1. 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 °C, on a moss and gravel substrate, mist them daily with reverse osmosis water, and feed them 3 times weekly with gut-loaded crickets.
2. 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.
3. 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.
   1. 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.

2. Testing for Bd Infection

1. Swabbing for Bd
   1. 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.
   2. 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 °C until extraction.
2. DNA Extraction and qPCR assay
   1. Extract genomic DNA from the swabs by adding 50 µL 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.
   2. Incubate the samples at 100 °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 °C until qPCR.
   3. Use quantitative PCR (qPCR) following Boyle et al. 2004 to analyze the infection load. Use the following modifications:
      1. Dilute DNA extraction samples 6:100 with double deionized water. Add 0.7 µL 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.

3. Inoculation

1. Culture Bd
   1. 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 °C for 40 min) and allow the broth to cool.
   2. 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 °C, then transfer the broth culture to TGHL agar plates.
   3. 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 °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.
   4. 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 °C for 5 – 7 d.
2. Flood plates for zoospore inoculation solution
   1. 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.
   2. 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.
   3. Estimate zoospore concentration following the manufacturer'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.
3. Inoculate animals
   1. 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.
      1. 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.
   2. 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.

4. Caspase 3/7 Assay

1. 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.
   1. 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 °C.
2. Extract proteins from frozen toe sample.
   1. Place samples in 100 µL 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 °C for 5 min. Collect supernatant to use in the assay.
3. Quantify protein concentration per sample using the Bradford assay.
   1. In a 384-well plate, mix 10 µL of protein extract with 10 µL 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.
4. 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.
   1. Before extracting the proteins from the sample for the caspase assay, photograph each toe at 40X magnification using an inverted light microscope.
   2. 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).
5. Perform the caspase 3/7 assay.
   1. In a 384-well luminescence plate, add 10 µL of commercially available caspase 3/7 reagent and 10 µL of protein extract. Run each sample in triplicate.
   2. 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.