2000 u eclipse microscope

To simultaneously record _cCa²⁺ and _mCa²⁺, LS8 cells and primary ameloblasts were loaded with 1 μM fluo4AM (ThermoFisher Scientific) and 4 μM rhod2AM (30 min at room temperature; ThermoFisher Scientific). To quantitate _cCa²⁺, cells were loaded with 1 μM fura2AM (1 h at room temperature; ThermoFisher Scientific) in Ca²⁺ containing Ringer’s solution [2 mM Ca₂Cl, 155 mM NaCl, 4.5 mM KCl, 3 mM MgCl2, 5 mM Na-Hepes, and 10 mM d-glucose (pH 7.4)]. To induce mitochondrial Ca²⁺ release 1 μM protonophore FCCP (Trifluoromethoxy carbonylcyanide phenylhydrazone; Sigma) (56 (link)) was added after 2 - 5 min in presence of 2 mM Ca²⁺. To stimulate ER Ca²⁺ release, ameloblasts were treated with 100 μM ATP in free Ca²⁺ ringer solution (EGTA 100 μM). SOCE activity was stimulated by pre-incubation with 2 μM thapsigargin (20 min; Sigma), placed in free Ca²⁺ ringer solution and then perfused with ringer solution containing 2 mM Ca²⁺. Fluorescence intensities were recorded every 3 to 5 s after excitation using the 20X magnification air objective on a Nikon 2000 U Eclipse microscope. The ratio F₃₄₀/F₃₈₀ of fura2AM values, the fluo4AM and rhod2AM fluorescence intensity, background corrected, were measured per each region of interest using Nikon ND software.

Measurements of [Ca²⁺]_i of single EO cells and LS8 cells were performed as described (6 (link), 22 (link)). Briefly, single cells were plated overnight on a round microscope cover glass in X-VIVO 15 medium supplemented with 10% FBS. Cells were loaded with 1 μM Fura-2 AM (Invitrogen) for 20 min at room temperature and washed in Ca²⁺-free Ringer solution [155 mM NaCl, 4.5 mM KCl, 3 mM MgCl₂, 5 mM Na-Hepes, and 10 mM d-glucose (pH 7.4)]. To increase cell purity in primary EO cells, we labeled fibroblasts using a PE-conjugated anti-CD90 antibody (1:500 dilution; BioLegend) and excluded the latter from further analysis. In EO and LS8 cells, ER store depletion was stimulated with either 1.25 μM thapsigargin (Sigma) or 0.5 μM ionomycin in Ca²⁺-free Ringer solution, followed by readdition of 2 mM extracellular Ca²⁺ Ringer solution to stimulate Ca²⁺ flux by SOCE. Fluorescence intensities at 510 nm were recorded every 5 s after excitation at 340 and 380 nm using a Nikon 2000 U Eclipse microscope or a FlexStation 3 plate reader. The ratio of F₃₄₀ and F₃₈₀ values correlating with [Ca²⁺]_i was calculated and graphed.