The authors would like to thank the staff of the Syracuse University Department of Physics workshop for their innovative assistance in matters relating to our system design.

1. Cleaning Cell Sonifier System
<ol>
	<li>Clean the union between the horn and converter using chloroform and a swab of cotton. Apply a light oil or mineral oil to the threads of the horn and converter to further remove buildup of metal shavings, rust, or oxidation.</li>
	<li>Use chloroform to remove the oil and any other contaminants from the union after wiping the threads with oil.</li>
</ol>
2. Reassembling the Cell Sonifier and Setting up the System&#160;
<ol>
	<li>Reattach the horn to the converter. To properly torque the system, remove the assembled converter and horn from the cup assembly by pulling the horn out of the bottom of the cup (Figure 2A). Place a slot wrench inside one of the three small holes near the top of the converter (Figure 2B). Then, position a torque-wrench fitted with a &#189; inch crow&#8217;s foot on the slotted part of the horn (Figure 2B). Tighten in standard clockwise direction until the torque meter reads 54.23 N.m (Figure 2C), the torque recommended by the manufacturer16. Fit the tightened converter and horn back into the cup, and mount the horn and converter to a ring stand in an upright position. Reattach the converter to the power supply before setting the system for experimental pulse dosing.</li>
	<li>Set the system for pulse dosing using 1 sec of sonication with 1 sec between pulses. Different dosing models may be used for other experimental conditions. Many variables, such as water volume and horn diameter would potentially require changes to this dosing. Using the aforementioned cell sample types, volume, and concentration, empirical data and observation have led to the formulation of this dosing regimen.</li>
	<li>Adjust the system to the desired amplitude. In this protocol, use 33% and 50% amplitude to prevent complete cell destruction, which would render the efficacy of sonosensitizers difficult or impossible to gauge. 
	NOTE: In this study the amplitudes are compared.</li>
</ol>
3. Assessing System Intensity and Function&#160;
<ol>
	<li>Hold the cavitation meter at 1.5 cm, which is the level of the sample sonication. Note the readings to assess that the system is running at an expected intensity. Using the current system and water levels, expect between 100-110 W/cm2 for 33% amplitude and 115-125 W/cm2 for 50% amplitude. Note the cavitation meter readings from the meter display which represents cavitation energy in W/cm2 directly. Take these readings at the surface of the water, and ensure no bubbles are present at the face of the meters probe. 
	NOTE: It is important to note that these procedures are done without the cap for access above the horn. They need not be repeated after every sonication. Rather, they should be performed only after cleaning, reassembling, and setting up the system.</li>
	<li>Place the hydrophone in a sample vial containing 12.5 ml of water in order to fully immerse the hydrophone sensor. Suspend the hydrophone using the ring stand.</li>
	<li>Attach the hydrophone to the input of the oscilloscope in order to assess waveform characteristics. Examine the oscilloscope for a clear sine wave, as aberrant waves can alter the frequency of the system from the specifications of the manufacturer. Both the cavitation meter and oscilloscope give a frequency reading, but only the oscilloscope will show aberrant waves that are indicative of a faulty system or improper assembly.</li>
</ol>
4. Preparation of Cells and Medium
<ol>
	<li>Prepare the medium by using 240 ml Iscove&#8217;s Modified Dubecco&#8217;s Medium (IMDM) and 50 ml fetal bovine serum. Do not thaw fetal bovine serum in a hot water bath, as rapid increases in temperature can lead to a compromise in serum stability. Combine 240 &#181;l gentamicin 50 mg/ml and 5 ml penicillin/ streptomycin 100x in a standard culture flask, and add to the medium. Store the medium at 4 &#176;C.</li>
	<li>Seed U937 cells at ~4 x 104 cells/ml in a 25 cm2 flask using the prepared medium. Assess cells for concentration and viability using a TC20 cell counter and trypan blue exclusion by placing 15-20 &#181;l of cells and 15-20 &#181;l trypan blue into a micro-centrifuge tube and mixing the contents. Pipette15-20 &#181;l of this mixture into a TC20 sampling slide, and initiate counts by placing the slide into the TC20 cell counter.</li>
	<li>Incubate cells at 37 &#176;C and 5% CO2, and check for cell viability using trypan blue exclusion and the TC20 cell counter. When the cells are at an appropriate concentration and/or have been treated with sonosensitizers for an appropriate time frame, transfer 3 ml of cells from the culture flask to a 20 ml glass scintillation vial. In this protocol, grow U937 cells for 48 hr, and then treat&#160;with 0.5, 1, or 5 mM Me&#946;CD for 30 min to sufficiently deplete the cholesterol of cells prior to sonication.</li>
</ol>
5. Cell Sonication
<ol>
	<li>Degas deionized, distilled water using a vacuum and Buchner flask. Transfer the water to the cup horn, and fill to a level of 15 mm above the top of the horn. The sonication process causes further degassing of the water for a period of a few minutes, and can lead to inconsistencies over the course of the experiment if the system is not run prior to sample sonication. Therefore, run the system for ~7 min prior to sample sonication.</li>
	<li>Attach the scintillation vial containing the cells to the holding device. Then, attach the holding device to the top of the cup horn and set to an elevation of 15 mm from the top of the horn (at the water surface using the sliding mechanism).</li>
	<li>The cells are typically sonicated using three 1 sec pulses of ultrasound, with 1 sec spacing in between each of these pulses. For this protocol, sonicate cells at 33% and 50% amplitude using the abovementioned pulse dosing.</li>
</ol>
6. Assessing Cells for Damage Post-sonication
<ol>
	<li>Assess the suspended cells for damage and viability using trypan blue and the TC20 cell counter. In addition, analyze the sample using a Z2 counter. For Z2 counter analysis, pipette a 100 &#181;l cell suspension into 20 ml isotonic saline (200:1 ratio). Prior to analysis, flush the aperture of the Z2 particle analyzer at least twice using isotonic saline. Place the Z2 sample in the counter holder, and raise the platform to the aperture before initiating counts.</li>
	<li>Acquire the data from the TC20 and Z2 counters using software provided by the manufacturer. Analyze these data for cell damage, viability, and the creation of cellular debris from sonication.</li>
</ol>
7. XTT Assay to Determine Cell Viability and Mitochondrial Activity of Treated U937 and THP1 Cells
<ol>
	<li>Prior to the XTT assays, grow U937 and THP1 cells under the same conditions. Pretreat cells with the same concentration range of Me&#946;CD for 30 min prior to sonication. Use the same ultrasound parameters as before except that cells are now sonicated using a range of 1-3 one sec pulses spaced one sec apart to develop a range of damage for the XTT kit to assess. 
	Note: Many of these steps are taken directly from the XTT kit manual and are only repeated for the convenience of interested laboratories.</li>
	<li>Collect cells by centrifugation at 200 x g for 10 min. Resuspend cell pellet in the growth medium previously described. Resuspend cells at ~1 x 105 cells/ml. Seed U937 cells at 100 &#181;l per well into a flat-bottom 96-well microtiter plate in triplicate, and then repeat for THP1 cells using a separate 96-well plate. Include 3 control wells containing 100 &#181;l of complete growth medium alone as blank absorbance readings. Then, incubate the inoculated plates for 24 hr.</li>
	<li>Defrost two aliquots of the XTT reagent and the activation reagent at 37 &#176;C prior to use. Swirl aliquots gently until clear solutions are obtained. Add 0.1 ml of the activation reagent to 5.0 ml of the XTT reagent, which forms enough activated XTT solution for one 96-well microtiter plate assay. Repeat the process for the other plate.</li>
	<li>Add 50 &#181;l of the activated-XTT solution to each well. Return the plate to the cell culture CO2 incubator for 2 hr. Shake the plate gently following the incubation period to evenly distribute the orange color in the positive wells. Measure the absorbance of the assay wells containing the cells and the blank background control wells at a wavelength between 450-500 nm wavelength using a microtiter plate reader.</li>
	<li>Either zero the microtiter plate reader using the blank control wells or subtract their average value from the specific results. Measure the absorbance of all the assay wells again at a wavelength between 630-690 nm and subtract the values from the 450-500 nm values obtained. Note: This second absorbance determination helps eliminate non-specific readings from the assay results. The XTT assay was repeated four times for both cell lines.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+shock+wave+lithotripsy:+a+comprehensive+review.">Optimizing shock wave lithotripsy: a comprehensive review.</a> Rev Urol. 15 (2), 49-60 (2013).</li><li>Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healing+sound:+the+use+of+ultrasound+in+drug+delivery+and+other+therapeutic+applications.">Healing sound: the use of ultrasound in drug delivery and other therapeutic applications.</a> Nat Rev Drug Discov. 4 (3), 255-260 (2005).</li><li>Cordeiro, E. R., Cathelineau, X., Th&#252;roff, S., Marberger, M., Crouzet, S., de la Rosette, J. 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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+focused+ultrasound+treatment+for+patients+with+unresectable+pancreatic+cancer.">High-intensity focused ultrasound treatment for patients with unresectable pancreatic cancer.</a> Hepatobiliary Pancreat Dis Int. 11 (6), 655-660 (2012).</li><li>Xiaoping, L., Leizhen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+of+high+intensity+focused+ultrasound+(HIFU)+for+pancreatic+cancer.">Advances of high intensity focused ultrasound (HIFU) for pancreatic cancer.</a> Int J Hyperthermia. 29 (7), 678-682 (2013).</li><li>Lukka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+focused+ultrasound+for+prostate+cancer:+a+systematic+review.">High-intensity focused ultrasound for prostate cancer: a systematic review.</a> Clin Oncol (R Coll Radiol). 23 (2), 117-127 (2011).</li><li>So, A. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-site+sonoporation+disrupts+actin+cytoskeleton+organization.">Single-site sonoporation disrupts actin cytoskeleton organization.</a> J R Soc Interface. 11 (95), 20140071 (2014).</li><li>Trendowski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+enlargement+of+leukemia+cells+using+cytoskeletal-directed+agents+and+cell+cycle+growth+control+parameters+to+induce+sensitivity+to+low+frequency+ultrasound.">Preferential enlargement of leukemia cells using cytoskeletal-directed agents and cell cycle growth control parameters to induce sensitivity to low frequency ultrasound.</a> Cancer Lett. In press, (2015).</li><li>Dumontet, C., Sikic, B. 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The authors declare that they have no competing financial interests.

To attain optimal results, special care should be taken to carefully position the sample and clean the converter-horn union. The placement of the sample in the horn is important for obtaining consistent cell destruction, as changing the distance from the horn will alter the acoustic foci, and therefore alter the energy the sample is exposed to. The acoustic energy within the cup horn can be mapped using the cavitation meter to find the position of maximum cavitation. In addition, the cavitation meter, along with the oscilloscope are vital for determining the sound intensity the cells are being exposed to, as well as the homogeneity of the waveform. Therefore, these instruments should be used to detect problems with the system, and help determine what troubleshooting may be needed to correct system instability.
As previously mentioned, the low frequency system may act to further degas the water throughout the experiment if not run for several minutes prior to sample sonication. This initial run must be performed to yield a relatively degassed sonication medium and thus consistent results during experiments. In addition, cells should not be sonicated at or near the maximum amplitude when assessing the efficacy of sonosensitizers, as the true extent of sensitization would be difficult to assess. Using 33% amplitude on the 40 kHz system is an ideal setting, as it produces notable damage, but provides sonosensitizers ample room to demonstrate their efficacy, as demonstrated with Me&#946;CD against U937 and THP1 cells (Figure 7). These data also confirm that Me&#946;CD sensitizes multiple leukemia lines to low frequency ultrasound in a dose dependent manner.
There have been a number of experiments done with higher frequency in the range of 0.75 MHz to 8 MHz showing evidence of intramembrane cavitation bubbles being generated through sonication17-19. However, questions still remain in regards to the exact mechanism of ultrasound-induced cell lysis18. We have shown a link between fluidizing the cytoskeleton and increased sonic sensitivity using low frequency ultrasound15, a phenomenon demonstrated by other laboratories20, 21. In addition, we have found that microfilament-disrupting agents such as cytochalasin B potentiate ultrasonic sensitivity in multiple leukemia lines, but not hHSCs or leukocytes22, suggesting that inhibition of actin polymerization may be a sonosensitizing mechanism of particular interest. We have also observed that vincristine, a microtubule-disrupting agent that inhibits tubulin polymerization23, 24, markedly increases the ultrasonic sensitivity of different leukemia types in vitro including acute myeloid leukemia, chronic myeloid leukemia, and acute lymphoid leukemia. By contrast, cytoskeletal-directed agents that stabilize cytoskeletal components (paclitaxel and jasplakinolide) appear to make cells resistant to sonication, reflected by lower rates of cell lysis22. Taken together, these data support the hypothesis that fluidizing the cytoskeletal components of neoplastic cells is indeed an important factor in increasing the efficacy of SDT25. The present study also demonstrates that cholesterol depletion may be another method by which to further potentiate the ultrasonic sensitivity of neoplastic cells, as Me&#946;CD-treated U937 cells are markedly sensitized to 40 kHz ultrasound.
While our sonication protocols have demonstrated marked antineoplastic activity in vitro, the current methodology is limited to work in culture and small vertebrate models that are able to fit in the vials used for sonication. We have shown that zebrafish can be safely sonicated using pulsed low frequency ultrasound (20 kHz), and that their tolerance to chemotherapeutic agents is quantitatively comparable to doses tolerated by murine models26, suggesting that tumor-bearing zebrafish may be used in preliminary investigations to assess the in vivo antineoplastic activity of these protocols. Nevertheless, administering chemotherapeutic agents prior to sonication of mammalian models has been reported in the MHz range1, and such protocols can likely be extended to incorporate low frequency ultrasound, as well as cholesterol-depleting and cytoskeletal-directed agents.
Potential clinical applications of this form of SDT may involve extracorporeal blood sonication in which antineoplastic agents are administered intravenously (i.v.) prior to the blood being removed for sonication25. This method removes potential sound barriers posed by human anatomy, and may be an effective way to damage leukemic blasts, as well as metastases from solid tumors. It is also possible that cholesterol-depleting and cytoskeletal-directed agents could be used in HIFU protocols that are already being examined in the clinic in an attempt to improve the efficacy of this treatment modality.
The methods described in the present study are capable of assessing the value of potential sonosensitizers, and further system refinement may enhance this utility. However, there are many variables to be considered when using such ultrasonic devises, including power supply quality, acoustic foci, and individual variation among converters. Therefore, future research will focus on visualizing the sonic waves and understanding their influence on results. SDT has shown to enhance cell lysis in vitro and may prove to be clinically viable if more in vivo data in mammalian models are acquired. Experiments examining other potentially exploitable characteristics of malignant cells, as well as various combined modalities involving multiple agents and ultrasound continue in our laboratory.

Ultrasound refers to any oscillating sound pressure wave with a frequency greater than the upper limit of human auditory capacities (~ 20 kHz)1. Low frequency ultrasound in the 20-60 kHz range has been utilized in the laboratory as a means of generating emulsions, preparing cellular samples for nucleic acid extraction, for tissue disruption, and for a variety of other tests. The utility of low frequency ultrasound has also been extended to the industrial setting for welding, cleaning various materials, and in materials processing. Commercially available ultrasound generators come in frequencies ranging from 18-60 kHz, and full-scale wattages from 100-1,200 W.
Although ultrasound has long been used in the clinical setting for diagnostic imaging, it has been applied as a therapeutic modality only recently. Ultrasound &#8805;1 MHz is capable of safely disrupting urinary calculi (kidney stones) and biliary calculi (stones in the gallbladder or in the liver) in patients to reduce symptoms2,3. This approach known as extracorporeal shockwave lithotripsy (ESWL) is now widely applied in the clinic (more than one million patients are treated annually with ESWL in the United States alone4), and offers a modality by which to non-invasively break up calculi with minimal collateral damage through the use of externally applied, focused, high intensity acoustic pulses2-4.
Due to the unique direct shearing forces, as well as cavitation bubbles generated by high intensity ultrasound, these methodologies have been examined in cancer therapy for the treatment of castration-resistant prostate carcinoma and pancreatic adenocarcinoma in an approach known as high intensity focused ultrasound (HIFU)5-8. In a manner very similar to ESWL, HIFU uses multiple ultrasound beams and focuses them on a selected focal area to generate temperatures of 60 &#176;C or higher through the use of acoustic energy, inducing coagulative necrosis in the targeted tissue5. Although other modalities of thermal ablation currently exist (radiofrequency ablation and microwave ablation), HIFU offers a distinct advantage over these methods in that it is the only non-invasive hyperthermic modality5. HIFU has attained mixed results in the clinic and is currently only available in clinical trials8-11. Nevertheless, the limited success it has achieved, and the very promising in vivo data acquired from preclinical mammalian models have demonstrated the potential of ultrasound in cancer therapy.
In an effort to improve HIFU, researchers have attempted to combine ultrasound with appropriate antineoplastic agents to generate a form of sonochemotherapy. Sonodynamic therapy (SDT) is a promising novel treatment modality that has demonstrated impressive antineoplastic activity in both in vitro and in vivo studies1. It has been shown that ultrasound preferentially damages malignant cells based on the size differential between such cells and those of normal histology1,5. SDT incorporates specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells. While therapeutic applications of SDT have been previously examined, ultrasonic systems used typically employ higher frequency ultrasound (&#8805;1 MHz), and the effects of low kHz frequency ultrasound has yet to be fully explored. Lower frequencies of ultrasound are often more proficient at producing inertial cavitation, a phenomenon that results in the destruction of cells due to the rapid collapsing of microbubbles, inducing physicochemical damage12-14. This difference in the generation of inertial cavitation between MHz and low kHz ultrasound has been attributed to the fact that lower wave frequencies enable microbubbles more time to grow by rectified diffusion in the expansion half cycle, consequently producing more violent collapses during the following compression half cycle12.
We have previously shown that U937 human monocytic leukemia cells are sensitive to low frequency ultrasound (23.5 kHz), and that this sensitivity can be markedly increased through the application of antineoplastic agents that perturb the cytoskeleton15. Further, we have demonstrated that cells are preferentially damaged based on size, with larger cells exhibiting higher ultrasonic sensitivity. In addition, normal human hematopoietic stem cells (hHSCs) and leukocytes at comparable cell sizes are much more resistant to sonication than their neoplastic counterparts15, tentatively suggesting that low frequency ultrasound may be used to preferentially damage malignant cells in the presence of normal tissue.
To further examine the unique properties of low frequency ultrasound for potential therapeutic use, we have developed cleaning and stabilization procedures to increase the efficacy and reliability of one of our current sonication systems, the Branson Model SLPe 150 W, 40 kHz Cell Disrupter, equipped with a 20 mm horn fitted into a 7.62 cm cup. In addition, we have been able to determine accurate sample cavitation energies, as well as consistent waveforms and amplitude within the 40 kHz range using a cavitation meter and oscilloscope with hydrophone. By refining and systematizing our protocols, we have been able to establish consistency in our experimental sonications, allowing us to quantitatively compare the sonic sensitivities of neoplastic and normal cells of different histogenetic lineages. Our protocol for the 40 kHz system is presented in extensive detail in order for interested laboratories to be capable of performing comparable experiments, and to evaluate our findings of the antineoplastic effects elicited by low frequency ultrasound. In addition, we examine the dose dependent effects of methyl-&#946;-cyclodextrin (Me&#946;CD; Figure 1), a cholesterol-depleting agent, on increasing the ultrasonic sensitivity of U937 and THP1 human monocytic leukemia cells.

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	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium w/ NaHCO3 &#38; 25mM Hepes&#160;</td>
			<td>Life Technologies</td>
			<td>12440079</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A2942&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x Solution&#160;</td>
			<td>Life Technologies</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12105</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson SLPe 40kHz Cell Disruptor with 3&#34; (25mm) Cuphorn</td>
			<td>Branson Ultrasonics</td>
			<td>101-063-726</td>
			<td>sonication device</td>
		</tr>
		<tr>
			<td>Brisk Heat SDC Benchtop Digital temperature Controler w/ 1000mL Beaker Heater</td>
			<td>Brisk Heat</td>
			<td>SDCJF1A-GBH1000-1</td>
			<td>heater used for temperature control</td>
		</tr>
		<tr>
			<td>Beckman-Coulter Z2 Cell Sizer with AccuComp&#174; Software</td>
			<td>Beckman-Coulter</td>
			<td>6605700</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>145-0102</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin 50mg/mL</td>
			<td>Sigma-Aldrich</td>
			<td>G1397&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL &#38; 25mL Vented Culture Flasks</td>
			<td>Fisher Scientific</td>
			<td>353082</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza L-Glutamine 200mM 0.85% NaCl</td>
			<td>Lonza</td>
			<td>17-605C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Seal-Rite 1.5 mL Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman-Coulter Accuvette ST 25mL Vials and caps</td>
			<td>Beckman-Coulter</td>
			<td>&#160;A35473</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuJet Pro Auto Pipet&#160;</td>
			<td>BrandTech Scientific</td>
			<td>26330</td>
			<td></td>
		</tr>
		<tr>
			<td>USA Scientific 10mL Disposable Serological Pipets</td>
			<td>USA Scientific</td>
			<td>1071-0810</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip One 100uL and 1000uL Filter Tips</td>
			<td>USA Scientific</td>
			<td>1120-1840, 1126-7810</td>
			<td></td>
		</tr>
		<tr>
			<td>100uL Micropipette&#160;</td>
			<td>Wheaton</td>
			<td>851164</td>
			<td></td>
		</tr>
		<tr>
			<td>1000uL Micropipette</td>
			<td>Wheaton</td>
			<td>851168</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad Dual Chamber Counting Slides</td>
			<td>Bio-Rad</td>
			<td>145-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Scientific Dual chamber water jacketed Incubator</td>
			<td>Forma Scientific</td>
			<td>3131</td>
			<td></td>
		</tr>
		<tr>
			<td>Tektronix&#160; DPO 2002B Digital Phosphor Oscilloscope</td>
			<td>Tektronix</td>
			<td>DPO2002B</td>
			<td>used to measure the ultrasonic waveform</td>
		</tr>
		<tr>
			<td>PPB MegaSonics Model PB-500 Ultrasonic Energy Meter</td>
			<td>PPB Megasonics</td>
			<td>PB-500&#160;</td>
			<td>used to assess the sound intensity in W/cm2</td>
		</tr>
		<tr>
			<td>Teledyne RESON TC4013-1 Hydrophone</td>
			<td>Teledyne</td>
			<td>TC4013-1&#160;</td>
			<td>connects to the oscilloscope&#160;</td>
		</tr>
		<tr>
			<td>Wheaton 250mL Flasks</td>
			<td>Sigma-Aldrich</td>
			<td>Z364827</td>
			<td></td>
		</tr>
		<tr>
			<td>20mL Glass Scintillation Vials&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Z190527</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman-Coulter Isotonic Saline Solution&#160;</td>
			<td>Beckman-Coulter</td>
			<td>N/A</td>
			<td>diluent for Z2 counter</td>
		</tr>
		<tr>
			<td>Chloroform 99%</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof Anhydrous&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>N/A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XTT Cell Proliferation Assay Kit</td>
			<td>ATCC</td>
			<td>30-1011K</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Microplate Reader</td>
			<td>Cole-Palmer</td>
			<td>&#160;EW-13055-54</td>
			<td></td>
		</tr>
		<tr>
			<td>U937 Human Monocytic Leukemia Cells</td>
			<td>ATCC</td>
			<td>CRL&#173;1593.2</td>
			<td></td>
		</tr>
		<tr>
			<td>THP1 Human Monocytic Leukemia Cells</td>
			<td>ATCC</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and quantitative analysis of pulsed low frequency ultrasound to determine the sonic sensitivity of untreated and treated neoplastic cells

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In addition, this method can be used in combination with specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells, an approach referred to as sonodynamic therapy (SDT). The methodology for generating and applying low frequency ultrasound in a preclinical in vitro setting is presented to demonstrate that reproducible cell destruction can be attained in order to examine and compare the effects of sonication on neoplastic and normal cells. This offers a means by which to reliably sonicate neoplastic cells at a level of consistency required for preclinical therapeutic assessment. In addition, the effects of cholesterol-depleting and cytoskeletal-directed agents on potentiating ultrasonic sensitivity in neoplastic cells are discussed in order to elaborate on mechanisms of action conducive to sonochemotherapeutic approaches.

System stability is paramount in obtaining repeatable and reliable results. The proper torque specification of 54.23 N.m achieved proper coupling of the transducer and sonotrode2, with the consistency of the waveform being assessed through the use of an oscilloscope and hydrophone (Figure 3A). Figure 3B depicts measurements inside the sample vial, and Figure 3C assesses stability within the cup horn. As expected, the amplitude was slightly reduced due to some energy being absorbed by the glass. However, the waveform was not perturbed or distorted in either panel B or C, which is necessary for reliable delivery of sonic energy to the sample. A waveform that does not appear as a clear singular sine wave indicates a faulty transducer or improper setup. This is expressed in Figure 3D which shows multiple aberrant waves, a clear absence of a sine wave, and a frequency reading not expected with a properly functioning system. The cavitation meter used to measure the cavitation energy generated (Figure 4) found the sound intensity to be 100-110 W/cm2 at 33% amplitude, and 125-130 W/cm2 at 50% amplitude.
Once the 40 kHz system was appropriately calibrated and assembled in its entirety (Figure 5), reliable cell destruction was readily attained, as determined by both the TC20 and Z2 counters (Figure 6). As expected, more extensive cell damage was observed in cell populations sonicated at 50%, rather than at 33% amplitude. Nevertheless, 33% amplitude is useful as a starting point to detect potential sonic sensitizing effects of administered agents, as it produces noticeable damage, but leaves room for more damage to be detected when agents are applied to assess their potentiation of ultrasonic sensitivity. This is demonstrated in Figure 7A, where the dose dependent effects of Me&#946;CD on potentiating the ultrasonic sensitivity of U937 cells are presented. Although non-sonicated Me&#946;CD-treated cells were very similar in viability to non-sonicated, untreated cells, the viability markedly dropped after three pulses of 1 sec 40 kHz ultrasound was applied. In addition, the decrease in viability after sonication appears to have been dose dependent, as 5 mM Me&#946;CD produced the largest drop in cell count, followed by 1 mM, and then 0.5 mM Me&#946;CD.
Similar effects on cell viability and mitochondrial activity were observed in both U937 and THP1 cells that were treated with varying concentrations of Me&#946;CD prior to sonication, as assessed with the XTT kit (Figure 7B). Although THP1 cells appear to be slightly more sonic resistant than U937 cells, both human leukemia lines are damaged in a dose dependent manner. Higher concentrations of Me&#946;CD and more pulses of ultrasound produced the lowest reduction of XTT to a soluble, brightly colored orange derivative for both U937 and THP1 cells, corresponding to lower cell viability and mitochondrial activity.
<img alt="Figure 1" src="/files/ftp_upload/53060/53060fig1.jpg" /> 
Figure 1.&#160;Molecular structure of methyl-&#946;-cyclodextrin. (A) Complete structure of methyl-&#946;-cyclodextrin (Me&#946;CD). (B) Me&#946;CD is a polymer of seven repeating units with a R group of either H or CH3. Molecular Formula: C56H98O35. Molecular Weight = 1331.36.
<img alt="Figure 2" src="/files/ftp_upload/53060/53060fig2.jpg" /> 
Figure 2.&#160;Properly torqueing the 40 kHz ultrasound system. (A) The horn and converter are removed from the cup prior to torqueing. (B) Position the slot wrench and torque wrench at their respective positions. (C) Apply 54.23 N.m of torque in a clockwise direction before reattaching the horn and converter to the cup.
<img alt="Figure 3" src="/files/ftp_upload/53060/53060fig3.jpg" /> 
Figure 3.&#160;Use of an oscilloscope to characterize the waveform of the ultrasonic system. (A) An oscilloscope with hydrophone can be used to evaluate amplitude and waveform consistency. (B) A recording taken with the cup directly above the horn at 1.5 cm and set at 33% amplitude. (C) The waveform pictured here is from a reading acquired when the hydrophone is placed within the 20 ml glass scintillation vial. The vial is positioned at 1.5 cm above the horn and again set at 33% amplitude. (D) An oscilloscope screenshot of a system with aberrant frequencies as would be expected with a faulty system or improper coupling of the horn converter union. <a href="https://www.jove.com/files/ftp_upload/53060/53060fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53060/53060fig4.jpg" /> 
Figure 4. Use of a cavitation meter to characterize the sound intensity of the ultrasonic system. The cavitation meter seen here is used to represent cavitation energy in W/cm2. This is a very important instrument for assessing consistency of energy with respect to the sample.
<img alt="Figure 5" src="/files/ftp_upload/53060/53060fig5.jpg" /> 
Figure 5. Key components of the 40 kHz ultrasound system. The ultrasonic system is shown complete with cup horn (A), heating apparatus (B), the converter (C), and sample positioning mechanism (D). This design allows for easy positioning of the sample (E) above the horn (F).
<img alt="Figure 6" src="/files/ftp_upload/53060/53060fig6.jpg" /> 
Figure 6. Effects of different levels of amplitude on the ultrasonic lysis of U937 cells using the TC20 and Z2 counters. Cells were sonicated with the 40 kHz cell dismembrator at 33% amplitude (100-110 W/cm2) or 50% amplitude (125-130 W/cm2) using three 1 sec pulses of ultrasound, with 1 sec spacing in between each of these pulses. &#160;(A) A screenshot from the Z2 counter software demonstrates the effect of sonication on U937 cells.&#160;(B) Data from the TC20 counter were compiled in a graph to demonstrate the reproducibility of the results. These data include the total and live cell counts, as well as the trypan blue assessed cell viability. Bars reflect the standard error of the mean (SEM) for 4 independent cell populations. <a href="https://www.jove.com/files/ftp_upload/53060/53060fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53060/53060fig7.jpg" /> 
Figure 7. Effects of methyl-&#946;-cyclodextrin on potentiating the ultrasonic sensitivity of U937 and THP1 cells. (A) U937 cells were treated with varying concentration of Me&#946;CD for 30 min prior to being sonicated with 40 kHz ultrasound (105 W/cm2) using three 1 sec pulses spaced 1 sec apart. Cell populations were grouped into &#8805;12 &#181;m and &#8805;17 &#181;m. (B) U937 or THP1 cells were again treated with varying concentrations of Me&#946;CD for 30 min prior to being sonicated with the 40 kHz system using 1-3 pulses of 1 sec ultrasound spaced 1 sec apart. Cell viability and mitochondrial activity were assessed with the XTT kit. 100 &#181;l of cells were seeded per well into a flat-bottom 96-well microtiter plate. The plate was incubated for 24 hr prior to the addition of XTT solution. Cells were then incubated for an additional 2 hr before the wavelength was read. Bars reflect SEM for 4 independent cell populations. <a href="https://www.jove.com/files/ftp_upload/53060/53060fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells at JoVE.c

Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

Selective damage of human leukemia cells can be achieved through a novel approach of applying low frequency ultrasound both with and without chemotherapeutic pretreatment of leukemic and normal hematopoietic cells.

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In ...

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Medicine

11.1K Views.  Syracuse University. Selective damage of human leukemia cells can be achieved through a novel approach of applying low frequency ultrasound both with and without chemotherapeutic pretreatment of leukemic and normal hematopoietic cells. 

Video: Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...