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

The overall goal of the following experiment is to assess the efficacy and reproducibility of pulse, low frequency ultrasound in damaging human leukemia cell populations in the absence or presence of a cholesterol depleting agent. This is achieved by cleaning the sonication system and evaluating its stability. The human leukemia cells are then sonicated in the absence and the presence of methyl beta cyclodextrin.

Next automated cell counters are used to assess cell viability and the effects of sonication and cholesterol depletion. On the mitochondrial activity of leukemia cells are evaluated by XTT assay. Ultimately, the data suggests that low frequency ultrasound may be used to damage malignant cells, which is potentiated by the use of a cholesterol depleting agent.

Our laboratory is interested in sonar namp therapy, an novel therapeutic modality that combines the anti-neoplastic effects of ultrasound with specialized agents to amplify the effects of son. Our protocol assesses the sonic sensitivity of both neoplastic and normal cells determine potential ano chemotherapeutic approaches that warrant further preclinical investigation. Generally, individuals new to this method may struggle with the equipment used to assess the stability of the so system as it can take some time to learn how to optimally apply those devices.

Visual demonstration of this method is critical as cleaning and stabilization procedures used to assess the sonication system are imperative for obtaining reproducible Results. Before beginning an experiment, use chloroform and A cotton swab to clean the union between the horn and converter. Next, apply a light oil to the threads of the horn and converter.

To further remove any metal shavings, rust, or oxidation buildup, then remove the oil and any other contaminants from the union with more chloroform and reattach the horn to the converter. To properly torque the system, pull the horn out of the bottom of the cup and place a slot wrench inside one of the three small holes near the top of the converter. Now, position a torque wrench fitted with a half inch crow's foot on the slotted part of the horn and tighten in the standard clockwise direction until the torque meter reads 39.99 foot pounds.

After tightening, fit the converter and horn back into the cup and mount the pieces to a rings stand in the upright position, reattach the converter to the power supply, and then set the system for pulse dosing. Using one second of sonication with one second between pulses, then adjust the system to the desired amplitudes to assess the intensity and function of the sauna Fire system. Begin by holding the cavitation meter at 1.5 centimeters, taking care that no bubbles are present at the face of the meter probe.

Read the cavitation meter screen to confirm that the system is running at the appropriate intensity. Next, fully immerse the hydrophone sensor in a sample vial containing 12.5 milliliters of water, and use the rings stand to suspend the hydrophone. To confirm the stability of the waveform characteristics, attach the hydrophone to the input of the oscilloscope and confirm the presence of a clear sine wave as aberrant waves can alter the Frequency of the system.

To prepare the cells for sonication First, seed them at four times 10 of the fourth cells per milliliter in a 25 square centimeter flask using freshly prepared medium. After determining the number of viable cells by triam blue exclusion, incubate the culture at 37 degrees Celsius and 5%carbon dioxide until the culture has reached the desired concentration. When the cells are ready for sonication, use a vacuum and bookner flask to degas a volume of deionized distilled water.

Then use the water to fill the cup horn up to 15 millimeters above the top of the horn. Run the system with the Degas water for about seven minutes. Then transfer three milliliters of cells into a glass 20 milliliter scintillation vial, and attach the vial to the holding device.

Next, attach the holding device to the top of the cup horn and use the sliding mechanism to set the holding device to an elevation of 15 millimeters from the top of the horn at the water surface. Now sonicate the cells using three one second pulses of ultrasound with a one second spacing between each of the pulses at 33 and 50%amplitude, assess the cell viability after sonication by triam blue exclusion. To assess the cell damage, flush the aperture of a Z two particle analyzer at least two times with isotonic saline.

Next, add 100 microliters of the sonicated cells into 20 milliliters of isotonic saline, and transfer the cell sample into the counter holder. Then raise the platform to the aperture and analyze the cell damage, viability, and debris according To the manufacturer's instructions. To assess the viability of sonicated cells By XTT assay, sonicate the cells using a range of one to three.

One second pulses spaced, one second apart to develop a range of damage for the XTT kit to assess, then spin down the cells and resuspend the pellets to one times 10 of the fifth cells per milliliter in freshly prepared growth medium. Next seed, 100 microliters of cells per well into individual flat. Bottom 96, well microtiter plates and triplicate, including three control wells containing 100 microliters of complete growth media alone for blank absorbance readings.

Incubate the cells for 24 hours on the morning of the assay, gently swirl aliquots of 37 degrees Celsius, warmed XTT and activation reagents until the solutions are clear. Then add 0.1 milliliters of the activation reagent to a five milliliter Eloqua of the XTT reagent. Next, add 50 microliters of the activated XTT solution to each well of the U 9 37 and THP one inoculated plates and return the cells to the cell culture incubator.

After two hours, shake the plates gently to evenly distribute the orange dye in the positive wells and measure the absorbence on a microtiter plate reader between 450 and 500 nanometers. Finally, measure the absorbence of all the assay wells again, between 630 And 690 nanometers. As expected, more Extensive cell damage is observed in cell population sonicated at the 50%than at the 33%amplitude.

The 33%amplitude, however, permits the detection of further damage when agents are applied. As in this experiment in which the dose dependent effects of the cholesterol depleting agent methyl beta cyclodextrin. On potentiating, the ultrasonic sensitivity of U 9 37 cells were evaluated.

Although the nons methyl beta cyclodextrin treated cells were similar in viability to the nons untreated cells, the viability markedly dropped after three pulses of one second, 40 kilohertz ultrasound were applied. Further, the decrease in viability after sonication appears to have been dose dependent as five millimolar of the cholesterol depleting agent produced the largest drop in cell count followed by one millimolar and 0.5 millimolar treatments with methyl beta cyclodextrin as assessed by XTT assay. Although the THP one cells appeared to be slightly more sonic resistant than the U 9 37 cells, both of the human leukemia cell lines were damaged in a dose dependent manner.

In addition, the coupling of higher concentrations of methyl beta cyclodextrin with a greater number of ultrasound pulses produced the lowest reduction of XTT to a soluble brightly colored orange derivative for both the U 9 37 and THP one cells, corresponding to a lower cell viability and mitochondrial activity For both cell lines. Following this procedure, other sonic Sensitizing agents such as cytoskeletal directed or reactive action species producing agents can be assessed for their potentiation of sonic sensitivity, thereby establishing novel therapeutic approaches that can be investigated for their plea clinical efficacy. While attempting this procedure, it's important to remember to appropriately organize the SON data acquired from the TC and Z two counters as they provide detailed information about the efficacy of the treatment protocol that can be lost.

If no logistical scheme is used After watching this video, you should have a good understanding of how to use low frequency ultrasound in combination with potential ano sensitizers in preclinical in vitro models to assess their potential therapeutic Utility.