I am a second year PhD student at Wayne State University looking to screen a library of photosensitizers (transition metal complexes) for activity with ultrasound irradiation in cell cultures (likely MCF-7A cells). I would ideally like to set this up in a 96-well plate to screen multiple compounds but I am unsure of the set up as previous literature has not been as helpful as I would hope. The instrument I have is 3 MHz with a 3.5 cm diameter probe (Model-Sonic 103, Preamonex). I am unfamiliar with soundwaves so I will briefly discuss what I know so far from previous literature.
Overall, 0.3 W cm–2, 3.0 MHz seems to be a fairly common treatment condition when combining compounds with cell cultures.
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This source clusters samples in a 2x2 square in the well plate and places the probe underneath the plate per cluster (see SI). Does a coupling agent need to be used and can this be done while keeping the plate sterile for continued incubation? Do the ultrasound waves pass through the plate to other clusters of wells (in a way that would significantly affect the other clusters)? Is there a way to evenly apply ultrasound stimulation evenly across the whole plate?
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This source works with nanoparticles and a 96-well plate, but details of the set up are not given. Unless I'm missing something?
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This source appears to place a sponge in degassed water on the head of the 35 mm probe. Could this be a valid option for the cluster of wells (2x2 square) mentioned above?
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For the purpose of transfection, a 6-well plate was put into an ultrasound water bath.
Ideally, I would like to use the probe (as opposed to a water bath) to keep the instrumentation consistent between in vitro and in vivo studies. As I mentioned, I'm very unfamiliar with sonication/ultrasound and its physics. Any assistance/advice on how to make an accurate and precise high throughput set up is appreciated.
Thank you in advance!
Setting up a high throughput system for ultrasound irradiation/sonication, especially for your specific application of screening photosensitizers in cell cultures, requires careful consideration of various factors. Given your current understanding and equipment, here are some steps and considerations to help you set up an accurate and precise high throughput system:
Coupling Agent and Sterility:
- A coupling agent is often used to facilitate the transmission of ultrasound waves from the probe to the sample. It helps minimize air gaps that can impede wave propagation. Sterile coupling gels or fluids can be used to maintain aseptic conditions for continued incubation.
- Plate Positioning:
- Based on the literature you mentioned, positioning the probe underneath the plate in a 2x2 square cluster seems to be a common approach. This can help focus the ultrasound energy on specific wells. The use of a coupling agent between the probe and the plate is crucial for effective transmission.
- Plate Material and Ultrasound Transmission:
- The choice of plate material can affect the transmission of ultrasound waves. Opt for plates that are compatible with ultrasound transmission, such as plastic or materials with low acoustic impedance. Glass plates might attenuate the ultrasound energy.
- Even Ultrasound Stimulation:
- Achieving even ultrasound stimulation across the entire plate can be challenging. The setup you mentioned with a probe underneath each cluster can help localize the energy, but it might not provide uniformity across the entire plate. Experimentation and optimization are key to finding the best setup for your specific needs.
- Impact on Adjacent Wells:
- Ultrasound waves can interact with neighboring wells. While some energy might pass through to adjacent clusters, the effect would likely be attenuated. However, it's important to monitor this potential interaction, especially if you're aiming for precise and controlled results.
- Probe Configuration:
- Since you're using a probe (as opposed to a water bath), consider whether modifying the probe configuration, such as using an array of smaller probes or optimizing the positioning, could help achieve more even energy distribution.
- Calibration and Dosimetry:
- Accurate dosimetry is essential for consistent results. Calibrate your ultrasound system to ensure that the intended energy levels are being delivered to the samples. This calibration can involve measuring the intensity, power, and frequency of the ultrasound waves at different points within the plate.
- Temperature Monitoring and Control:
- Ultrasound irradiation can lead to localized heating. Monitor and control the temperature during experiments to ensure that any effects observed are due to sonication and not temperature-induced changes.
- Safety Considerations:
- Ultrasound can induce cavitation, which may affect cells and compounds differently. Carefully consider safety protocols and ensure that the chosen parameters do not cause harm to the cells or compromise the integrity of your experimental setup.
- Collaboration and Consultation:
- Collaborate with experts in the field of ultrasound and sonication. Consult with colleagues, mentors, or specialists who have experience in similar setups to gain insights and advice.
Remember that achieving an accurate and precise high throughput system requires iterative experimentation and optimization. As you become more familiar with ultrasound physics and its interaction with your samples, you'll be better equipped to fine-tune your setup for optimal results.
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