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

Area of Science:

• Cell Biology
• Immunology

Background:

• Thapsigargin is a known inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase.
• Bivalent cations, such as Ca2+, play critical roles in cellular signaling pathways.

Purpose of the Study:

• To investigate the dynamics of thapsigargin-induced bivalent-cation entry into antigen-specific B cells.
• To determine the origin of intranuclear calcium (Ca2+) signals.

Main Methods:

• Confocal fluorescence microscopy using fluo-3 and fura-2 dyes.
• Stimulation of B cells with thapsigargin in the presence and absence of external Ca2+.
• Use of Ba2+ and Mn2+ as Ca2+ channel probes.

Main Results:

• Thapsigargin stimulation led to Ca2+ accumulation in both the cytoplasm and nucleus of B cells.
• In the absence of external Ca2+, nuclear and cytoplasmic Ca2+ levels returned to baseline.
• Subsequent addition of external Ca2+ re-established elevated Ca2+ levels in both compartments.
• Ba2+ and Mn2+ quenched fluorescence, indicating entry through Ca2+ channels and supporting cytoplasmic origin of nuclear Ca2+.

Conclusions:

• Thapsigargin-induced nuclear Ca2+ signals in B cells are likely derived from the cytoplasm, not internal nuclear stores.
• Bivalent cation entry into B cells involves pathways accessible to Ca2+, Ba2+, and Mn2+.