Simple method of aequorin loading into platelets using dimethyl sulfoxide.

Area of Science:

• Biochemistry
• Cell Biology
• Hematology

Background:

• Investigating intracellular calcium (Ca2+) dynamics is crucial for understanding platelet activation.
• Aequorin, a photoprotein, can be used to measure Ca2+ concentrations but requires efficient loading into cells.
• Dimethyl sulfoxide (DMSO) has been explored as a method for enhancing cellular permeability.

Purpose of the Study:

• To evaluate the efficacy and safety of using DMSO for aequorin loading into human platelets.
• To determine if DMSO treatment affects platelet function and integrity.
• To assess the feasibility of using DMSO-loaded platelets for luminescence-based Ca2+ measurements.

Main Methods:

• Washed human platelets were incubated with aequorin and varying concentrations of DMSO.
• Platelet membrane integrity was assessed by lactate dehydrogenase (LDH) leakage.
• Platelet aggregation, ATP release, and ultrastructure were examined.
• Luminescence signals were measured upon stimulation with agonists like collagen, thrombin, and A23187.

Main Results:

• A 6% DMSO concentration, added stepwise over 2 minutes, maximized aequorin penetration into platelets.
• DMSO treatment caused negligible membrane damage and preserved platelet ultrastructure and function (aggregation, ATP release).
• Platelets loaded with aequorin via DMSO emitted sufficient luminescence signals upon stimulation, enabling Ca2+ concentration monitoring.

Conclusions:

• The DMSO method provides a simple, rapid (2 min at room temperature), and effective means to load aequorin into human platelets.
• This technique allows for the study of intracellular Ca2+ changes during platelet activation without compromising platelet viability or function.
• DMSO-mediated aequorin loading is a valuable tool for investigating platelet calcium signaling pathways.