Current in vitro sonication experiments show immense variability in experimental set-ups and methods used. As a result, there is uncertainty in the ultrasound field parameters experienced by sonicated samples, poor reproducibility of these experiments and thus reduced scientific value of the results obtained. The scope of this narrative review is to briefly describe mechanisms of action of ultrasound, list the most frequently used experimental set-ups and focus on a description of factors influencing the outcomes and reproducibility of these experiments. The factors assessed include: proper reporting of ultrasound exposure parameters, experimental geometry, coupling medium quality, influence of culture vessels, formation of standing waves, motion/rotation of the sonicated sample and the characteristics of the sample itself. In the discussion we describe pros and cons of particular exposure geometries and factors, and make a few recommendations as to how to increase the reproducibility and validity of the experiments performed.

Department of Medical Biophysics Faculty of Medicine and Dentistry Palacky University Olomouc Hnevotinska 3 Olomouc 775 15 Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc Hnevotinska 5 Olomouc 779 00 Czech Republic

Joint Department of Physics and Cancer Research UK Cancer Imaging Centre Division of Radiotherapy and Imaging The Institute of Cancer Research London and The Royal Marsden NHS Foundation Trust Sutton London SM2 5PT United Kingdom

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