Calcium Imaging in LAD2 Mast Cells

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.