Confocal fluorescence microscopy for studying thapsigargin-induced bivalent-cation entry into B cells.

We studied thapsigargin-induced bivalent-cation entry into antigen-specific B cells (TP67.21) with a confocal fluorescence microscope. Confocal fluorescence images of fluo-3-loaded B cells showed that thapsigargin-stimulated Ca2+ signals were transferred not only to the cytoplasm but also to the nucleus. In the absence of external Ca2+ ions, the free Ca2+ concentrations both in the cytosol and in the nucleus declined to basal levels by 5 min after addition of thapsigargin. However, subsequent addition of Ca2+ in the external medium made the fluo-3 (fura-2) fluorescence intensity rise, reflecting the fact that Ca2+ accumulated again in the nucleus as well as in the cytoplasm. Then, we added Ba2+ and Mn2+ instead of Ca2+, because Ba2+ and Mn2+ are known to enter via Ca2+ channels. The addition of Ba2+ and Mn2+ in the external medium quenched the fluo-3 fluorescence both in the nucleus and in the cytoplasm of B cells. This suggested the possibility that the increase in intranuclear Ca2+ after thapsigargin stimulation may come from the cytoplasm, not from the nuclear stores.