A non-disruptive technique for loading calcium buffers and indicators into cells

Area of Science:

• Cell biology
• Biochemistry
• Physiology

Background:

• Current methods for intracellular calcium measurement/buffering are invasive or perturb cell integrity.
• Existing techniques are limited to specific cell types or require complex procedures.
• Previous loading methods for calcium chelators have significant drawbacks.

Purpose of the Study:

• To develop a simple and effective technique for loading calcium chelators into intact cells.
• To overcome the limitations of existing methods for intracellular calcium buffering.
• To demonstrate the applicability of the new method across different cell types.

Main Methods:

• Developed a method to temporarily increase cell membrane permeability to calcium chelators.
• Utilized esterifying groups to mask chelator carboxylates, enabling cell entry.
• Hydrolysis of ester groups inside the cell regenerates active chelators and traps them cytoplasmically.
• Demonstrated the technique on red blood cells, mast cells, and lymphocytes.

Main Results:

• Successfully loaded Ca2+-selective chelators into intact cells in suspension.
• The method avoids cell lysis and significant perturbation of cytoplasmic constituents.
• The technique is versatile and applicable to various mammalian cell types.
• Intracellular calcium buffering was achieved without compromising cell viability.

Conclusions:

• The described technique offers a non-invasive and efficient way to buffer intracellular calcium.
• This method expands the possibilities for studying calcium dynamics in diverse cell populations.
• The approach provides a valuable tool for cell physiology research.