Imaging of total intracellular calcium and calcium influx and efflux in individual resting and stimulated tumor mast cells using ion microscopy

Ion microscopy was employed to investigate intracellular total calcium concentrations and calcium influx, and efflux in resting and antigen-stimulated tumor mast cells (RBL-2H3 cells). The nucleus, a perinuclear region which included the Golgi apparatus (Golgi region), and the remaining cytoplasm were spatially resolved with the Cameca IMS-3f ion microscope in cryogenically prepared cells. In resting cells the nucleus contained about 0.60 mM, the Golgi region about 1.2 mM, and the remaining cytoplasm about 1.0 mM total calcium. Antigen stimulation of rat basophilic leukemia cells resulted in a significant loading of calcium in all three cellular compartments. Antigen stimulation in the absence of extracellular calcium resulted in a significant loss of total calcium from all three intracellular compartments. Influx and efflux of calcium were measured simultaneously in resting and stimulated cells by using stable 44Ca in the extracellular solution, and by imaging mass 40 to determine the native intracellular calcium (40Ca) and mass 44 to localize the 44Ca that entered the cell from extracellular solution. After a 10-min incubation, 0.240 fmol of the total calcium per cell had been replaced with 44Ca, which amounts to about 33% of the total cell calcium. If antigen was present during this incubation there was an additional loss of 0.229 fmol of 40Ca and an added gain of 0.476 fmol of 44Ca per cell, which corresponds to a net increase in total intracellular calcium of 0.247 fmol.