Calcium imaging in primary neurons

For the measurement of store Ca²⁺ release and SOCE, cells were washed with M1 medium²¹, following which they were loaded with 2.5 μM of Fluo-4 acetoxymethylester (AM; Life Technologies) with 0.002% Pluronic-F-127 (Sigma-Aldrich) in M1 medium for 30 min in the dark. Following dye loading, cells were washed in M1 and placed in nominally ‘0’ Ca²⁺ M1 (containing 12 nM-free Ca²⁺) supplemented with 0.5 mM EGTA and imaged for Ca²⁺ changes. Imaging and quantification were performed as described previously²¹. Images were acquired every 15 s (for passive store depletion induced SOCE) or every 10 s (for CCh-stimulated SOCE) for a total duration of 10 min (600 s) on a Nikon TE2000 inverted wide-field microscope equipped with × 60/1.4 numerical aperture (NA; oil) objective lens, using the 488-nm excitation and 520-nm emission filter sets. Store depletion was induced by adding 10 μM TG (Life Technologies) or 20 μM Carbamoylcholine chloride (CCh; Sigma) after the first frame of acquisition. CCh-containing medium was exchanged with ‘0 Ca’ medium after 200 s, and the cells were imaged for another 100 s before Ca²⁺ add-back. CaCl₂ was added after the 20th frame of acquisition (or 300 s). CCh measurements were performed with overexpression of muscarinic acetylcholine receptor in neurons of all genotypes. For the measurement of resting cytosolic Ca²⁺, a previously described protocol²¹ was followed. In brief, cells were incubated with 5 μM Indo-1 AM and 0.002% Pluronic F-127 in M1 media for 45 min at room temperature in the dark. Cells were washed twice with M1 before and after dye incubation and finally covered with M1 for imaging. Data acquisition was performed by 365-nm excitation and 410/485 dual-emission filter sets at 5-s interval for five frames, after which 10 μM ionomycin (Calbiochem, USA) was added to obtain the maximum florescence intensity and imaged for 10 more frames. EGTA (1 mM) and 0.01% Triton-X-100 were added after the 10th frame to obtain the minimum florescence intensity for each cell. Image acquisition for basal calcium measurements was performed on an Olympus IX81-ZDC2 inverted wide-field microscope equipped with focus drift compensating and × 20/0.5 NA (air), × 60/1.35 NA (oil) and × 100/1.4 NA (oil) objective lenses. Excitation of fluorescent Ca²⁺ indicator dye was performed using specific wavelength illumination from a halogen arc lamp with a TILL Polychrome 5000 monochromator (TILL Photonics, Graefelfing, Germany) for variable bandwidth and intensity. Emitted light was detected through band-pass filter sets (Chroma, Brattleboro, VT). Image acquisition was performed using the Andor iXON 897E EMCCD camera and AndoriQ 2.4.2 imaging software. The time-lapse acquisition mode of the software was used to follow fluorescence changes over time.