Flow cytometric detection of intracellular platelet antigens

Exocytosis following platelet activation leads to translocation of CD62P (P-selectin), CD63, and thrombospondin from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. We report the detectability of these molecules preformed--prior to platelet activation--inside the cytoplasm of resting platelets. Two different methods are described, using either methanol or the Fix&Perm kit for cell membrane permeabilization. In addition, interleukin(IL-)1 alpha is shown to be present in platelet cytoplasm after methanol treatment but not after permeabilization using Fix&Perm. Whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining only. Our data demonstrate the feasibility of the methods described for the detection of intracellular platelet molecules.