Encyclopedia of Experiments: Cancer Research
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Transcript
- To begin, seed the human leukemia cells in a freshly prepared culture medium and incubate for the desired period. After incubation, stain a small amount of this cell suspension with trypan blue and assess cell viability using a TC20 cell counter slide. Dead cells are permeable and take up the negatively charged dye, while viable cells are impermeable.
Next, add methyl beta cyclodextrin to the culture flask to sufficiently deplete the cell's cholesterol, making them susceptible to ultrasound. Incubate the cells for the desired period. Fill the cell sonication system with deionized water and degas it for a few minutes to avoid vacuum bubbles. Transfer the desired amount of cells in the scintillation vial and attach the vial to the cell sonication system's holding device.
Place the holding device on top of the horn and set the desired elevation from the horn's top. Set required pulses for the desired duration to sonicate the cells. Add trypan blue to the cell suspension and place it in the TC20 cell counter to analyze cell viability and damage. Methyl beta cyclodextrin-treated leukemia cells, when exposed to low frequency ultrasound, show significant cell lysis.
In the following protocol, we will study cell sonication and damage assessment of human leukemia cells.
- To prepare the cells for sonication, first seed them at 4 times 10 to the fourth cells per milliliter in a 25 square centimeter flask using freshly prepared medium. After determining the number of viable cells by trypan 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 Buchner flask to degas a volume of ionized distilled water. Then use the water to fill the cup horn up to 15 millimeter above the top of the horn.
Run the system with the degassed water for about seven minutes, then transfer 3 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 seconds 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 trypan blue exclusion. To assess the cell damage, flush the aperture of a Z2 particle analyzer at least 2 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.
