Research data of Calcium Imaging after electrical stimulation

<p>For intracellular calcium ions (Ca²⁺) mobilization analysis by Calcium Imaging, a human osteoblast-like cell line MG-63 (ATCC &reg;, CRL1427&trade;) 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 &deg;C in a humidified atmosphere (5% CO₂) in Dulbecco&#39;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 <em>in vitro</em> 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&deg;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 &mu;M) in slightly hypotonic 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES) buffer [1-3] shaking at 37&deg;C. After incubation for 40 min, cells were centrifuged and then resuspended in 250 &micro;l of isotonic HEPES. In a 12-well plate, 750 &micro;l of isotonic HEPES was placed. Then the stained suspended cells were added, and immediately electrical&nbsp;stimulation&nbsp;by using IonOptix chamber&nbsp;was started under the LSM780 (Carl Zeiss). After 5 min of adhesion phase, the global calcium signal of the attaching&nbsp;cells was visualized using the inverted LSM 780 with a C Apochromat 40&times; 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 &quot;time series&quot; mode. The first time series with electrical stimulation included 150 cycles each 2 s. After this time series&nbsp;(total electrical stimulation of 10 min), the electrical stimulation was stopped and a second time series without electrical stimulation&nbsp;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].</p> <p>[1] Staehlke S, Koertge A, Nebe B (2015) Intracellular calcium dynamics dependent on defined microtopographical features of titanium. Biomaterials 46: 48&ndash;57.</p> <p>[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.</p> <p>[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.</p>