Low Frequency Ultrasound Application: A Method to Determine the Effect of Sonication on Human Leukemia Cells

1. Preparation of Cells and Medium

1. Prepare the medium by using 240 ml Iscove’s Modified Dubecco’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 µl 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°C.
2. 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 µl of cells and 15-20 µl trypan blue into a micro-centrifuge tube and mixing the contents. Pipette15-20 µl of this mixture into a TC20 sampling slide, and initiate counts by placing the slide into the TC20 cell counter.
3. Incubate cells at 37 °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 with 0.5, 1, or 5 mM MeβCD for 30 min to sufficiently deplete the cholesterol of cells prior to sonication.

2. Cell Sonication

1. 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.
2. 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).
3. 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.

3. Assessing Cells for Damage Post-sonication

1. 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 µl 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.
2. 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.