Simple method of aequorin loading into platelets using dimethyl sulfoxide

Abstract

To investigate changes in Ca2+ concentrations in platelet cytoplasm during the activation, aequorin was loaded into platelets with an incubation of dimethyl sulfoxide (DMSO) in platelet suspension. When washed human platelets (about 5 X 10(9) platelets/microliter) were incubated with 10 microM aequorin and 6% DMSO (final concentrations) for 2 min at room temperature, a part of aequorin penetrated through the platelet membrane into the cytoplasm. The leakage of LDH was very slight indicating membrane damage by DMSO treatment being negligible. The platelet membrane and cytoplasmic components preserved their normal features under electron microscope. Platelet aggregation and ATP release were not affected by the incubation. The amount of permeated aequorin was the largest when DMSO was added stepwisely 6 times for 2 min to reach 6% final concentration. Though about 1/5,000 to 1/10,000 of added aequorin penetrated into platelets, the platelets emitted enough luminescent signals by additions of collagen, thrombin and A 23187. The DMSO method is very simple and can save the incubation time (only 2 min at room temperature) to load aequorin into platelets.