For intracellular calcium ions (Ca2+) mobilization analysis by Calcium Imaging, a human osteoblast-like cell line MG-63 (ATCC ®, CRL1427™) were used [1-3]. The MG-63 cell line has similar characteristics in terms of morphological behavior, adhesion and signaling properties as primary human osteoblasts [3]. Cells were cultured at 37 °C in a humidified atmosphere (5% CO2) in Dulbecco's modified eagle medium (DMEM; Gibco) containing 10% fetal calf serum (FCS; PAA Laboratories) and 1% antibiotic (gentamicin, Ratiopharm GmbH) [1-3]. Cells in the near-confluent state (70-80%) were used for the corresponding in vitro experiments. Therefore, cells were washed with PBS, trypsinized with 0.05% trypsin/0.02% EDTA (PAA) for 5 min and then treated with medium to stop the reaction. After centrifugation, 2x106 cells / 2ml were incubated shaking in complete medium at 37°C for a complete independent experiment. For the method, 2.5x105 cells were stained per electrical stimulation setup (non-stimulated, 1 V 7.9 Hz, 1 V 20 Hz, 5 V 7.9 Hz or 5 V 20 Hz). First, a wash step was performed with PBS, and after centrifugation, sedimented cells were stained with a membrane-permeable calcium indicator fluo-3/AM (Life Technologies Corporation; 5 μM) in slightly hypotonic 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES) buffer [1-3] shaking at 37°C. After incubation for 40 min, cells were centrifuged and then resuspended in 250 µl of isotonic HEPES. In a 12-well plate, 750 µl of isotonic HEPES was placed. Then the stained suspended cells were added, and immediately electrical stimulation by using IonOptix chamber was started under the LSM780 (Carl Zeiss). After 5 min of adhesion phase, the global calcium signal of the attaching cells was visualized using the inverted LSM 780 with a C Apochromat 40× water immersion objective. Fluo-3/AM dye was excited with the argon ion laser at 488 nm (emission at 515 nm). A full frame (512 x 512 pixels) at maximum pinhole aperture was evaluated using Zen2011 (black edition) software (Carl Zeiss) and "time series" mode. The first time series with electrical stimulation included 150 cycles each 2 s. After this time series (total electrical stimulation of 10 min), the electrical stimulation was stopped and a second time series without electrical stimulation was started. This second time series comprised 240 cycles every 2 s. After the 90th cycle of these series, fluo-3/AM stained cells were treated with 10 mM adenosine-triphosphate (ATP; SERVA). Mean fluorescence intensity of cells was evaluated using Zen2012 software (blue edition) and Mean ROI mode for defined areas of individual single cells [1-3]. [1] Staehlke S, Koertge A, Nebe B (2015) Intracellular calcium dynamics dependent on defined microtopographical features of titanium. Biomaterials 46: 48–57. [2] Staehlke S, Rebl H, Finke B, Mueller P, Gruening M, Nebe JB (2018) Enhanced calcium ion mobilization in osteoblasts on amino group containing plasma polymer nanolayer. Cell Biosci 8: 22. [3] Staehlke S, Rebl H, Nebe B (2019) Phenotypic stability of the human MG-63 osteoblastic cell line at different passages. Cell Biol Int 43:22-32.