## 1 Introduction  
Ultrasound has been proposed for an extensive variety of therapies with different modalities and intensities for producing either thermal or non-thermal effects. The low-intensity pulsed ultrasound (LIPUS) is a type of non-thermal ultrasound modality with intensities lower than 3 W/cm\({}^{2}\) applied in a pulsed regime that has been proposed for multiple medical applications [1, 2, 3, 4, 5, 6, 7]. Interestingly, those therapeutic applications range from tissue healing and cell survival stimulation to cell death induction (cancer therapy), in which comparable acoustic treatment conditions are used for producing opposite effects. For instance, ultrasonic parameters for cell apoptosis induction in cancer therapy [1, 2, 8, 9] are quite similar to those used for induction of increased survival under basal [3, 10] and stressing conditions [6]. These discrepant outcomes make the protocol designing for cell models that are not yet studied difficult.  
Moreover, there are diverse cell treatment setups that have been used with miscellaneous results. One of the most used setups for in vitro cell stimulation places the transducer below the cell culture plate with ultrasonic gel as a coupling media [1; 11]. Using this setup permits a mechanically stable configuration, with the disadvantage of increased standing waves in the culture media and, consequently, a high variability of results [12]. In another configuration, the culture plate is situated on the surface of a water tank above the planar transducer, which is separated from the dish at a certain distance [2; 10]; this arrangement has shown to have reduced standing waves and give more reproducible results [12]. However, other experimental configurations in which the cells are placed in a closed chamber immersed in a water tank [13; 14], or where the transducer is inserted into the cell culture media for cell stimulation from above [7; 15] have been scarcely used. Acoustic pressure measurements complemented with computational models suggest that those discrepancies in treatment setups originate a high variability of results and a low level of comparison between studies [12].  
Differences in stimulation protocols increase the variability in outcomes observed for in vitro stimulated cell cultures. Some authors made use of a single ultrasonic treatment that ranges from seconds to 20 min in duration [1; 7; 8; 10] while others fractionate the ultrasonic dose in three short treatments separated by a lapse of an ultrasonic recess [4; 16]. Moreover, the ultrasound stimulation can be applied once a day during two or more days [11; 17; 18; 19], or every other day [14]. Furthermore, the ultrasound can be applied using planar or focused transducers with different acoustic intensities, duty factors (DF), and pulse repetition frequencies (PRF) that increase the number of studied variables [20].  
Therefore, the development of an in vitro cell stimulation protocol with LIPUS should account for different types of variables; for instance, acoustic intensity, DF, PRF, setup configuration, time of exposure per session, and number of sessions per day/week. Those parameters can be tested in terms of the viability of each cell type to analyze according to the expected ultrasonic effects. In this paper, the viability analysis of a retinal glial cell line, MIO-M1, treated with LIPUS is presented. LIPUS was applied from the bottom of the culture dish using two setups commonly used for ultrasonic treatments of adherent cells. The viability was determined for different combinations of acoustic intensities, DFs and treatment durations. For validating the setup, the ultrasound transmission through the culture dish was measured; a finite element model of ultrasound and heat propagation in the culture well was analyzed. The reduction of standing waves using small relative inclinations between the transducer and the dish is proposed.