Plan apochromat 63x oil immersion objective

Samples for immunofluorescence analysis were prepared as described previously [23 (link)]. Fixation of cells with methanol or 4% paraformaldehyde were performed when primary antibody reactivity demanded, and gave the same results; however tubular profiles were best preserved in cells fixed with paraformaldehyde. Wide-field fluorescence microscopy images were acquired with an Axiovert 200M microscope equipped with a PlanApochromat 63x oil immersion objective (NA 1.4; Carl Zeiss), using SlideBook acquisition software (Intelligent Imaging Innovations, Denver, CO) without deconvolution of the images, or with an AxioObserver.D1 microscope equipped with a PlanApo 63x oil immersion objective (NA 1.4), and an AxioCam MRm digital camera (Carl Zeiss), using similar settings as described previously [23 (link)]. Laser scanning confocal microscopy images were acquired with an Olympus FluoView FV1000 scanning unit fitted on an inverted Olympus IX81 microscope equipped with a PlanApo 60x oil immersion objective (NA 1.4; Olympus, Melville, NY), using similar settings as described previously [24 (link)]. For quantification of phenotypes, two hundred cells were counted for each experiment.

The Ca²⁺ mobilization process in MG-63s was specified with respect to the adenosine 5′-triphosphate (ATP) concentration for cell stimulation, Ca²⁺ origin and the presence of ATP receptors as provided in the Supplementary Material. The basal Ca²⁺ signal of MG-63s on Ti and PPAAm controls was validated by immunofluorescence. Therefore, 50,000 cells/cm² were stained after 1 h with the Ca²⁺ indicator fluo-3 (5 μM, Life Technologies Corporation, Eugene, OR, United States) (Staehlke et al., 2015 (link), 2018 (link)), Hoechst 33342 (1:1000, Life Technologies Corporation) and for images after 24 h additionally with 20 μl BacMam 2.0 reagent (CellLight^TM actin-RFP, Life Technologies Corporation) at 37°C in isotonic 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) (Staehlke et al., 2015 (link), 2018 (link)). Confocal laser scanning microscopy (LSM780, Carl Zeiss, Jena, Germany) was carried out with a Plan-Apochromat 63x oil immersion objective (Carl Zeiss, 1.40. Oil DIC M27) and the ZEN software (ZEISS efficient navigation, ZEN 2011 SP4, black edition, Carl Zeiss).