Fluo 4 am fluorescent dye

Calcium mobilization in LAD2 cells was followed by fluorimetric analysis of cytoplasmic-free calcium with Fluo-4 AM fluorescent dye (Molecular Probes, Invitrogen) as described elsewhere [36] (link). Briefly, 0.2×10⁶ cells/point were loaded with 5 mM Fluo-4-AM for 30 minutes at 37°C in the dark, washed twice with Tyrode’s buffer, and resuspended. To measure calcium influx in the absence of extracellular calcium, cells were washed and resuspended with Tyrode’s buffer without calcium. Fluorimetric measurements were by a Modulus II Microplate Multimode Reader (Turner Biosystems, Promega, CA), according to the manufacturer’s instructions. After defining basal conditions the stimuli was added (time 0) and fluorimetric measures were done 10 more minutes. Each point was done by triplicate.

Fluo-4 AM fluorescent dye (Molecular Probes, Thermo Fisher Scientific, Rochester, NY, United States) was used for intracellular Ca²⁺ measurements. Cells were washed once with pre-warmed ACSF and then incubated with 2.5 μM fluo-4 AM for 20 min at 37°C. Then, cells were rinsed in order to incubate with ACSF for another 20 min, in turn allowing further de-esterification of the dye. Images were captured under confocal microscopy (Leica Microsystems, Buffalo Grove, IL, United States). The excitatory wavelength was 488 nm, and emissions were collected in the 505–515 nm range. To account for potential variation in dye loading among axons or experiments, a standard procedure was applied with non-ratiometric indicators, in which self-ratios were taken (F/F₀) between the measured fluorescence (F) and the initial fluorescence (F₀). Background fluorescence subtraction was accomplished by continuously sampling three areas in the field that had no axons in them for the duration of the experiment. Since fluo-4 AM is a non-ratiometric dye, we used CellTracker Red (CTR), a cytoplasmic marker, as a reference dye to avoid volumetric effects. In these experiments, 2.5 μM fluo-4 AM was used in tandem with 3 μM CTR. For CTR, the excitatory wavelength was 594 nm, and emissions were collected within the range of 600–610 nm.