In vivo imaging was performed using the IVIS Shared Resource at the University of Colorado Cancer Center (supported by grant P30-CA046934). Flow cytometry was performed at the Flow Cytometry Shared Resource, University of Colorado Cancer Center (supported by grant P30CA046934). This work was supported in part by the National Institutes of Health (RO1CA137078 to DKG). ABLS is a Fellow of the Pediatric Scientist Development Program, supported by grants from the American Academy of Pediatrics, the American Pediatric Society, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (K12-HD000850).

All experiments involving animals followed the regulatory standards approved by the University of Colorado Institutional Animal Care and Use Committee. The demonstrated protocol was approved by the University of Colorado Institutional Animal Care and Use Committee.
1. Phospho-protein Western Blot
<ol>
	<li>Preparation of lysates
	<ol>
		<li>Culture cell lines expressing the receptor tyrosine kinase of interest. Harvest cells and plate 3-5 x 106 cells/sample in 400 &#956;l growth medium in a 48-well tissue culture dish. Incubate at 37 &#176;C in 5% CO2 for 2-3 hr.</li>
		<li>Add 100 &#956;l of 5x TKI solution in growth medium and incubate for 60 min.</li>
		<li>Prepare lysis buffer with 50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, and protease inhibitors. If cultures will not be treated with pervanadate prior to harvest, add phosphatase inhibitors (1 mM sodium orthovanadate and 0.1 mM sodium molybdate) to lysis buffer.</li>
		<li>Prepare pervanadate (PV) phosphatase inhibitor solution immediately before use. Prepare a 0.3% solution of hydrogen peroxide (CAUTION) in cold phosphate-buffered saline (PBS) in a dark tube on ice (1:100 dilution of a 30% stock solution). Boil an aliquot of 0.2 M sodium orthovanadate (OV, CAUTION) stock solution for 5 min. Dilute 1:10 in PBS to make 20 mM OV solution at room temperature (RT). Mix 0.3% hydrogen peroxide and 20 mM OV 1:1 and incubate in the dark at RT for 20 min.</li>
		<li>Dilute PV into complete medium (60 &#956;l PV + 940 &#956;l medium). Add 100 &#956;l of diluted PV/well. Incubate at 37 &#176;C in 5% CO2 for 3 min and then place plate on ice.</li>
		<li>Harvest the cells in cold microfuge tubes at 2,500 rpm for 5 min. Aspirate supernatant and resuspend cells in 120 &#956;l of lysis buffer. Incubate on ice for 15 min. Clarify the lysate in a cold microfuge at 6,000 rpm for 3 min. Transfer supernatant to a fresh cold tube. Store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Immunoprecipitation
	<ol>
		<li>Spin down Protein G sepharose beads in microfuge at 1,000 rpm for 1 min. Remove supernatant and wash beads 2x with 1 ml cold PBS. Add an equal volume of PBS to make a 50% slurry. Add primary antibody and 20 &#956;l 50% Protein G beads to each sample. Incubate for 2 hr - O/N at 4 &#176;C on a rotator.</li>
		<li>Pulse down beads in a cold microfuge at 9,000 rpm. Remove supernatant and wash beads 2x with 150 &#956;l cold lysis buffer. Spin in cold microfuge at 1,000 rpm for 1 min. Remove supernatant and resuspend pellet in 20 &#956;l 2X SDS Sample Buffer with 2-mercaptoethanol (CAUTION). Use immediately or store at -80 &#176;C.</li>
		<li>Boil samples for 5 min and run on an SDS-PAGE tris-glycine gel. Transfer the proteins to a nitrocellulose membrane and detect phospho-protein by western blot.</li>
		<li>Rinse blot with water and then incubate in 2% SDS, 62.5 mM Tris (pH 6.8), 0.1 M &#946;-mercaptoethanol (CAUTION) for 30 min at 50 &#176;C. Rinse 2x with water and then wash for 60-90 min in 5-6 changes of TBS-T to remove stripping solution.</li>
		<li>Detect total-protein by western blot.</li>
	</ol>
	</li>
</ol>
2. Methylcellulose Assay
<ol>
	<li>Aliquot 4 ml of methylcellulose human base medium into 50 ml conical tubes using a sterile large bore blunt end needle. (Note: Methylcellulose can be aliquoted and stored at -20 &#176;C. Thaw at RT O/N prior to use.)</li>
	<li>Add 500 &#956;l of 10x TKI or vehicle in growth medium to 4 ml of methylcellulose and vortex mixture for 10 sec.</li>
	<li>Harvest and count cells. Prepare a cell suspension in growth medium in a concentration, which is 10x higher than the final cell concentration. Add 500 &#956;l cell suspension to methylcellulose mixture. Vortex for 10 sec and let sit for 10 min to remove bubbles.</li>
	<li>Draw up 4 ml of methylcellulose mixture using a 5 ml syringe and sterile large bore blunt end needle. Avoid drawing up bubbles. Dispense 1 ml of the methylcellulose mixture into the center of three 35 mm tissue culture plates. Swirl the dishes until the bottom is completely covered with methylcellulose.</li>
	<li>For each methylcellulose plate, prepare a 10 cm sterile tissue culture plate with two additional 35 mm tissue culture plates filled with sterile water to ensure a humid environment. Place cultures in water jacketed incubator at 37 &#176;C in 5% CO2 for 10-14 days.</li>
	<li>Count colonies after 1-2 weeks. Colonies should be more than 20 cells and not overlapping. Colonies can be counted using an inverted microscope equipped with a microscope stage with a grid or using an automated colony counter. Changes in colony size or morphology in response to treatment with TKI should also be evaluated. To improve detection by colony counter, stain colonies by adding 60-100 &#956;l of (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide solution (MTT, 5 mg/ml in H2O, store at -20 &#176;C, protect from light, CAUTION) drop-wise over the surface of each plate and incubating for 8 hr at 37 &#176;C in 5% CO2.</li>
</ol>
3. Soft Agar Assay
<ol>
	<li>Prepare 1% noble agar for bottom layer, 0.7% noble agar for top layer and 2x growth medium. Equilibrate 2x growth medium in a 42 &#176;C water bath. Heat 1% and 0.7% noble agar in a microwave (approximately 1 min/100 ml) and equilibrate in a 42 &#176;C water bath. Mix equal parts of 1% agar and 2x medium and separately 0.7% agar and 2x medium in 50 ml conicals. Plan 10 ml of soft agar mixture/six-well plate for each layer.</li>
	<li>Label 6-well tissue culture plates. Fill each well with 1.5 ml 1% soft agar mixture. Avoid bubbles during plating. Dry plates with lids off in sterile hood for 30 min.</li>
	<li>Count cells. Prepare a cell suspension in growth medium in a concentration, which is 500x higher than the final cell concentration. Add cell suspension to 0.7% agar mixture, mix well and dispense 1.5 ml of the agar cell mixture on the top of the 1% agar layer. Let the top layer solidify for 30 min.</li>
	<li>Prepare 1x growth medium containing TKI or vehicle control. Add 2 ml of medium with desired concentration of TKI on top of the top agar layer.</li>
	<li>Incubate cultures for 10 days at 37 &#176;C in 5% CO2. Remove and replace the overlaying medium every 3 days.</li>
	<li>When colonies are visible to the naked eye, aspirate overlaying medium and add 200 &#956;l nitro-tetrazolium blue solution (1 mg/ml NBT in H2O, store at 4 &#176;C, protect from light, CAUTION). Return to incubator for 24-48 hr. Move to 4 &#176;C for at least 2 hr prior to counting. Stained plates can be stored at 4 &#176;C for one month. Count colonies using a microscope or an automated colony counter.</li>
</ol>
4. Evaluation of Phospho-protein Inhibition In vivo
<ol>
	<li>Collect cells growing in log phase by centrifugation in a table top centrifuge at 1,500 rpm for 5 min. Wash the cells once with sterile PBS and resuspend in PBS at an appropriate concentration (typically 1-5 x 107 cells/ml). Transplant cells in a volume of 100 &#956;l into 6-12 week old NSG mice by intravenous injection into the tail vein using a 28 G insulin syringe. Place the animal in a restrainer, heat the tail in a beaker of warm water to dilate the veins, and clean the tail with a chlorhexidine swab prior to injection. After injection, apply pressure to the injection site using sterile gauze until bleeding stops. Transplant at least 3 animals/group. Maintain the mice on water containing 1 mg/ml gentamycin sulfate.</li>
	<li>Fourteen days post-transplant, treat animals with TKI or vehicle only and harvest samples for analysis of target inhibition. Weigh animals and treat with an appropriate dose (mg/kg of body weight) of TKI or vehicle. Administer treatment in a volume of 5 ml/kg body weight by intraperitoneal (i.p.) injection or 10 ml/kg body weight by oral gavage. For i.p. injection, restrain the mouse manually and inject in the lower right quadrant of the abdomen using a 29 G needle. For oral gavage, restrain the mouse manually and hold the animal upright. Place the gavage tube in the mouth over the tongue and into the back of the mouth. Apply slight pressure to pass the tube through the esophagus and into the stomach. Depress the syringe plunger to administer treatment. If the plunger does not depress easily the gavage needle should be removed and repositioned. Prepare TKI formulations immediately before use.</li>
	<li> If pervanadate treatment is desired, prepare PV solution 20 min before harvest as described above (step 1.1.4) and dilute into medium with 1 &#956;M MgCl2 and 100 U/ml DNase (120 &#956;l PV + 880 &#956;l medium). Note: PV is stable for at least 1 hr after dilution.</li>
	<li>Sacrifice animals by CO2 narcosis at the appropriate time(s) post-treatment. Dissect femurs by removing fur, skin and muscle from the entire leg so that the bones are completely exposed. Remove the femurs by clipping with dissecting scissors just below the hip joint and again above the knee joint. Transfer to a Petri dish and flush bone marrow with 1 ml cold PBS or diluted PV solution using a syringe and a 27 G needle. If treating with PV, incubate at RT in the dark for 10 min.</li>
	<li>Prepare lysates and analyze phospho-protein and total-protein levels as indicated above (steps 1.1.6-1.2.5).</li>
	<li>Quantitate relative phospho-protein and total-protein levels for each sample using densitometry. Calculate phospho-protein/total-protein relative to the average phospho-protein/total-protein value for vehicle-treated animals.</li>
</ol>
5. Evaluation of Anti-leukemia Activity of TKIs in ALL Xenograft Models
<ol>
	<li>If necessary, expose mice to 200 cGy of radiation in a RS-2000 irradiator one day prior to transplant. Transplant mice with leukemia cell lines as described above (step 4.1). Transplant at least 6 mice per group. Identify mice by ear punch. Alternatively, a black marker may be used to identify mice with hash marks on their tails. Add enrichment to cages to reduce the potential for cage mate aggression during the study.</li>
	<li>Treat mice with a TKI or vehicle only as described above (step 4.2). Monitor the health status of mice daily via physical examination and weight determination. Expected symptoms of leukemia (reduced activity level, weight loss, hind limb paralysis, and eye infections). Weight loss more than 10%-15% is usually associated with the death of the mouse within two days and is typically a surrogate endpoint for death in accordance with standard IACUC requirements.</li>
	<li>Monitor engraftment and development of leukemia via an in vivo bioluminescence imaging system up to 2x weekly. Prepare a 200x D-luciferin stock solution (30 mg/ml) in sterile water. Mix gently by inversion until D-luciferin is completely dissolved. Use immediately, or aliquot and freeze at -20 &#176;C. Prior to imaging using an in vivo bioluminescence imaging system, thaw aliquots of D-luciferin at RT and dilute 1:2 in PBS to a final concentration of 15 mg/ml and filter sterilize through a 0.2 &#956;m filter.</li>
	<li>Anesthetize the mice before imaging with 1.5% inhaled isoflurane in oxygen. Monitor sufficient anesthesia, characterized by muscle relaxation, loss of movement, and loss of response to toe-pinch stimulation.</li>
	<li>Immediately before imaging, administer 150 mg/kg body weight D-luciferin by i.p. injection (10 &#956;l of 15 mg/ml luciferin/g of body weight).</li>
	<li>Use an in vivo bioluminescence imaging system to capture 2 series of images with sequential 30, 60 and 90 sec exposures and a final image with a 120 sec exposure. After imaging, return mice to cages and monitor for recovery from anesthesia. Depending on the intensity of bioluminescence, exposure times can be varied. Optimal exposure times should be predetermined for a given model and kept consistent such that the signal intensities for individual time points are comparable across the entire study.</li>
	<li>Determine bioluminescence intensity for each mouse using Living Image 3.2 acquisition and analysis software. Determine total flux values measured in photons/second by drawing regions of interest (ROI) of identical size over each mouse.</li>
	<li>Sacrifice mice by CO2 exposure and cervical dislocation if moribund, exhibiting paralysis, in the event of greater than 15% weight loss, or at the end of the study. Dissect mice and record observations (e.g. enlargement of spleen, liver, or lymph nodes; pale bone marrow). Dissect femurs and flush bone marrow with cold PBS using a 27 G needle.</li>
	<li>Collect cells in a microfuge at 2,500 rpm for 5 min. Resuspend cells in PBS containing 2% FBS and incubate for 5 min at RT. Collect cells and stain with 20 mg/ml DAPI (4,6-Diamidino-2-phenylindole dihydrochloride, 1:1,000) and FITC-conjugated &#945;-hCD45 (1:100) or mouse IgG1 isotype control antibody (1:100). Evaluate leukemia engraftment using flow cytometry. Gate on the viable population (DAPI negative) and determine percentage of CD45+ cells (% bone marrow blasts).</li>
</ol>

<ol>
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The authors declare that they have no competing financial interests.

This manuscript describes an effective strategy for evaluation of novel tyrosine kinase inhibitors in the treatment of acute leukemia. Using this approach, biochemical and anti-leukemia activities are evaluated first in cell-based assays in vitro and then in xenograft models in vivo. Immunoblot analysis was successfully utilized to demonstrate inhibition of the target tyrosine kinase in leukemia cells after treatment with TKIs and to directly compare the potency of multiple compounds in cells. In the pr...

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children1,2. The overall survival rate for pediatric B-lineage ALL (B-ALL) is approximately 85%, but specific biological subtypes, including T-lineage ALL (T-ALL), have still poorer prognosis even with current therapeutic protocols. Further treatment of relapsed ALL remains a challenge3. Although the majority of adult patients with acute leukemia achieve a remission with up-front chemotherapy, many patients still suffer relapse4. Current chemotherapeutic regimens in the treatment of acute leukemia are known to cause toxicity-associated short- and long-term side effects. Therefore, less toxic therapies that specifically target cancer cells with minimal effect on normal tissues are greatly needed. In recent years, emphasis has been placed on the development of novel, molecularly-targeted agents with specificity for cancer cells, often utilizing iterative chemistry to generate multiple active compounds which must then be compared and prioritized5. This manuscript describes an efficient strategy for pre-clinical evaluation of TKIs for the treatment of acute leukemia, which can be used for evaluation of a single compound or for direct comparison of multiple compounds in order to facilitate drug development.
The method presented here consists of four steps. First the biochemical (1) and anti-leukemia (2) activities of the compound(s) are evaluated in cell culture, then inhibition of the target is confirmed in animal models (3), and finally therapeutic efficacy of the TKI(s) is determined in orthotopic leukemia xenograft models (4). For these studies, it is important to choose relevant cell lines, which are representatives of the most common biologic subtypes. Cell lines should be selected, which both, express the target of interest and lack the target of interest, to investigate whether biologic effects are mediated by inhibition of the target. This is particularly relevant for the development of small molecule inhibitors, which have off-target effects that may be important for anti-tumor activity. It is also necessary to choose a cell line that is dependent on the target for functional effects such as proliferation or survival. Preliminary target validation studies (outside the scope of this article) using RNA interference or other specific means to inhibit the target can be used to identify target-dependent cell lines. It is also desirable to choose cell lines that can form murine xenografts, such that cell culture results can be more directly translated to in vivo experiments.
For evaluation of biochemical activity mediated by TKIs in leukemia cells, a decrease in receptor phosphorylation can be used as an indicator of target inhibition. Western blot analysis or ELISA assays can be employed, depending on the availability and specificity of antibodies. If antibodies with sufficient specificity for the target are available, ELISA assays are preferable as they are more quantitative and efficient. In cases where antibodies with sufficient specificity for ELISA are not available, western blot analysis may be necessary. In this case, immunoprecipitation of a large amount of lysate can be useful for detection of targets that are in low abundance. This approach is particularly relevant for measurement of phospho-proteins, which may have a short half-life to allow for rapid changes in signaling in response to environmental stimuli. Some phosphorylated proteins are extremely labile, most likely as a result of complex formation with phosphatases. For robust and consistent detection of these phosphorylated proteins, it may also be possible to treat cells with pervanadate, an irreversible protein-tyrosine phosphatase inhibitor6, to stabilize the phospho-protein prior to preparation of whole cell lysates.
To determine whether biochemical activity results in anti-tumor effects, target-dependent biological processes can be monitored in cell-based experiments. For leukemia cell lines, anti-leukemia activity mediated by TKIs can be assessed using colony formation assays performed in methylcellulose or soft agar7. Soft agar may be preferred as this is a solid medium that is more amenable to manipulation if repeated treatment with a TKI is necessary. While many acute myloid leukemia (AML) cell lines will form colonies in soft agar, most ALL cell lines will only form colonies in methylcellulose, which is a semi-solid medium. Although it is possible to refresh medium and/or TKIs in methylcellulose cultures, only small volumes can be used and with limited frequency. Similarly, it is more difficult to stain colonies without disrupting them in methylcellulose. Preliminary studies should define the ability of appropriate cell lines to form colonies in methylcellulose and/or soft agar and the optimal density of cells in culture such that colonies are non-overlapping and in sufficient number to obtain statistically relevant data (usually 50-200 colonies per 35 mm plate).
While in vitro assays are robust and cost-effective, and have fewer ethical implications than whole animal experiments, advancement of therapeutic compounds requires proof of efficacy and safety in animal models. For in vivo studies, human acute leukemia cell lines can be orthotopically transplanted into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and TKIs can be easily administered by injection or oral gavage. Some cell lines may require exposure of NSG mice to a low cell line-dependent dose of radiation in order for xenografts to establish, and in this instance mice may not tolerate oral gavage without a 5-10 day recovery period post-irradiation. This manuscript describes generation of B-ALL and T-ALL xenografts using specific cell lines (697 and Jurkat) as examples but xenografts may be established in NSG mice using a wide variety of cell lines. In the event that other cell lines are more applicable, the requirement for irradiation, optimal number of cells to transplant, and timing of disease onset and progression should be determined experimentally. Ideally, these models will have complete penetrance (every animal transplanted develops leukemia), consistent kinetics (leukemia progresses similarly in all animals), and a reasonable treatment window (ideally 20-30 days between initiation of treatment and removal from study due to disease). The number of cells transplanted can be increased to improve penetrance and kinetic consistency or decreased to improve the treatment window if necessary.
To determine if TKIs mediate target inhibition in vivo, samples are collected from mice with leukemia xenografts after treatment with TKI or vehicle only. Ideally, the dose and schedule of administration for these experiments are guided by pharmacokinetic studies, which can often be performed by commercial laboratories and are outside the scope of this article. If pharmacokinetic data are available, the concentration of compound required for effective target inhibition in cell culture and the maximum serum concentration following a single dose of TKI can be used to define a starting dose for animal studies. Pharmacokinetic studies can also inform the timing of sample collection post-treatment for pharmacodynamic studies and the route of administration. Inhibition of the target can be assessed in any affected organ but tissues that are most easily collected and processed are preferable. Most acute leukemia cell lines establish in the liver, bone marrow, spleen, peripheral blood, and the central nervous system, although the specific organs affected and the extent of engraftment in these organs varies between models. The protocol presented here assesses phospho-protein inhibition in bone marrow using western blot analysis, but solid organs may be easier to consistently harvest and require minimal or no processing prior to freezing, allowing less opportunity for degradation of phospho-proteins during sample collection and processing. Immunohistochemistry can also be used to evaluate solid tumors or organs affected by leukemia.
Finally, the therapeutic efficacy of the TKI(s) is determined in orthotopic leukemia xenograft models. For these studies, the timing of treatment initiation can be varied, such that disease is more or less established. Treatment may begin immediately after transplant for initial studies and then be delayed in subsequent studies until significant disease burden is detected to more closely approximate a diagnostic treatment model. Ideally, these animal models also have the capacity for non-invasive measurement of disease burden. We have optimized methods for introduction of the firefly luciferase gene into leukemia cell lines using virus-like particles, allowing for non-invasive, longitudinal analysis of disease onset and progression and assessment of disease burden in bone marrow and solid organs. Critical to this approach is the use of monoclonal luciferase-tagged cell lines to prevent variability in development of luciferase-expressing leukemia associated with the use of polyclonal cell lines and is unrelated to treatment with a TKI8.
Taken together, these steps can be used for evaluation of a single TKI or for direct comparison and ranking of multiple TKIs. While the protocols presented here focus on development of TKIs, these methods can be adapted to other targets and considerations for assay development are described. Thus, this strategy may be more broadly applicable to the pre-clinical evaluation of molecularly-targeted agents for treatment of acute leukemia.

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			<td style="width:167px;"> </td>
			<td style="width:233px;"> </td>
			<td style="width:223px;">Reagent/Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Hydrogen Peroxide</td>
			<td style="width:167px;">MP Biomedicals</td>
			<td style="width:233px;">#02194057</td>
			<td style="width:223px;">GHS05, GHS07, H302-H318</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Sodium Orthovanadate</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#S6508</td>
			<td style="width:223px;">GHS07, H302+ H312+H332 </td>
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		<tr height="63">
			<td height="63" style="height:63px;width:443px;">2-Mercaptoethanol </td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#M7522</td>
			<td style="width:223px;">GHS05, GHS06, GHS08, GHS09, H301 + H331-H310-H315-H317-H318-H373-H410 </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">ColonyGel Human Base Medium </td>
			<td style="width:167px;">ReachBio</td>
			<td style="width:233px;">#1101</td>
			<td style="width:223px;"> </td>
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			<td height="42" style="height:42px;width:443px;">3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#M5655</td>
			<td style="width:223px;">GHS07, GHS08, H315-H319-H335-H341 </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Difco Noble Agar </td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:233px;">#214883</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Nitrotetrazolium Blue Chloride</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#N6639</td>
			<td style="width:223px;">GHS07, H302 </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">D-Luciferin Firefly, Potassium Salt</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:233px;">#122796</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">4&#39;,6- Diamidino-2-phenylindole dihydochloride</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#D9542</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">FITC CD45 </td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#347463</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">FITC Mouse IgG1 Isotype control</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#51-35404X-2</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Gentamycin Sulfate</td>
			<td style="width:167px;">Sparhawk</td>
			<td style="width:233px;">#NDC58005-633-04</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Protease Inhibitors </td>
			<td style="width:167px;">Roche </td>
			<td style="width:233px;">#11836153001</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">DNase</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#D4263 </td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Protein G Beads</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:233px;">#10-1242</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Isofluran</td>
			<td style="width:167px;">VETONE</td>
			<td style="width:233px;">#NDC13985-030-60</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;"> </td>
			<td style="width:167px;"> </td>
			<td style="width:233px;"> </td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;"> </td>
			<td style="width:167px;"> </td>
			<td style="width:233px;"> </td>
			<td style="width:223px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Cell culture dishes, diam. 35 mm &#215; H 10 mm</td>
			<td style="width:167px;">Nunclon</td>
			<td style="width:233px;">#D7804-500EA</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Cell culture dishes, diam. 100 mm x H 20 mm </td>
			<td style="width:167px;">Nunclon</td>
			<td style="width:233px;">#D8429-1CS</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">6-well plates</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#353046</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">14 gauge x 4 inch blunt-end needles</td>
			<td style="width:167px;">Cadence science</td>
			<td style="width:233px;">#7956</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">5 ml syringe with luer-lok</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#309646</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">GelCount automated colony counter</td>
			<td style="width:167px;">Oxford Optronix</td>
			<td style="width:233px;"> </td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">In vivo bioluminescence imaging system</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:233px;">#IVIS200</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Scout pro portable balances, scale</td>
			<td style="width:167px;">Ohaus</td>
			<td style="width:233px;">#SP202</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:443px;">Broome style rodent restrainer</td>
			<td style="width:167px;">Plas-labs</td>
			<td style="width:233px;">#551-BSRR</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ear punch, punch diameter: 2 mm</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#24210-02</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Chlorhexidine swabs, Prevantics</td>
			<td style="width:167px;">PDI</td>
			<td style="width:233px;">#B10800</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Insulin syringe 1 mL (40 Units) 29 G x 1/2</td>
			<td style="width:167px;">Monoject </td>
			<td style="width:233px;">#8881500042</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:443px;">Plastic feeding needles for rodents (disposable) 20 ga x 38 mm, sterile</td>
			<td style="width:167px;">Instech</td>
			<td style="width:233px;">#FTP-20-38</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">1 mL Luer-Lok disposable syringe</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#309628</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:443px;">Lo-Dose U-100 insulin syringe with 28 G x &#189;, permanently attached needle</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#329465</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Extra fine bonn scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#14084-08</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Student fine scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#91460-11</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Moria ultra fine forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#11370-40</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Extra fine graefe forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#11150-10</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Scalpel handle</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#10003-12</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Scalpel blades</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#10011-00</td>
			<td style="width:223px;"> </td>
		</tr>
	</tbody>
</table>

pre-clinical evaluation of tyrosine kinase inhibitors for treatment of acute leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

The assays presented here evaluate the biochemical and functional effects mediated by TKIs and can be used to rank novel compounds based on the degree of target inhibition in vitro and in vivo, reduction of colony formation, and delay in leukemogenesis in NSG mice transplanted with luciferase-tagged leukemia cells.
Immunoblot analysis was utilized to determine inhibition of the active phosphorylated form of the target protein in leukemia cells following treatment with TKIs. T...

Watch this Scientific Journal Video about Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia at JoVE.com

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

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18.1K Views.  University of Colorado Anschutz Medical Campus. Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Video: Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...