Thapsigargin

Thapsigargin (Acros Organics) was dissolved in dimethyl sulfoxide (DMSO) and added to NGM plates to a final concentration of 3 μg/ml, as reported previously (Zwaal et al., 2001 (link)), with the modification that Thapsigargin was added directly to the medium rather than supplemented on the surface.

HeLa cells were loaded with 4 µM Fura-2 AM (Molecular Probes, Thermo Fisher Scientific) in DMEM, and incubated for 45 min at 25 °C^{40 (link),41 (link)}. Then the cells were washed twice and incubated at room temperature in Ca²⁺-free buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 0.5 mM EGTA, 10 mM glucose in 10 mM HEPES-KOH, pH 7.35). Where indicated, HeLa cells were treated with siRNA for 96 h prior to Ca²⁺ imaging, and were treated with 1 µM Thapsigargin (Molecular Probes, Thermo Fisher Scientific). Ratiometric measurements were conducted for 7.5 or 12.5 min using an iMIC microscope and the polychromator V (Till Photonics), with alternating excitation at 340 and 380 nm and measurement of the fluorescence emitted at 510 nm. The microscope was equipped with a Fluar M27 lens with ×20 magnification and 0.75 numerical aperture (Carl Zeiss), and an iXon^EM + camera (Andor Technology). Images containing 50–55 cells/frame were sampled every 3 s using TILLvisION software (Till Photonics). Fura-2 signals were recorded as the F340/F380 ratio, where F340 and F380 correspond to the background-subtracted fluorescence intensities at 340 and 380 nm, respectively. Cytosolic [Ca²⁺] was estimated from ratio measurements using an established calibration method^{54 (link)}. Data were analyzed using Excel 2007. P-values were determined using unpaired t-tests.