Fluorescent Labeling of Platelets with Polyanionic Fluorescein Derivatives

OBJECTIVE: To investigate whether polyanionic fluorescein derivatives are capable of labeling resting and activated hamster and human platelets. STUDY DESIGN: 5,6-Carboxyfluorescein and calcein were incubated with resting and convulxin-activated hamster and human platelets in the 0-3.4 microM and 0-2.5 microM final concentration range, respectively, and assayed by flow cytometry and confocal microscopy. RESULTS: Resting and activated platelets of both species could be labeled by both fluorophores and quantified by flow cytometry. The fluorescence emission intensity and the fraction of labeled platelets increased linearly with final fluorophore concentration. Examination of labeled platelets by confocal microscopy revealed that 5,6-carboxyfluorescein and calcein colocalized with the platelet glycocalyx and that the fluorophores were often sequestered by the cells. The latter manifested itself by compartmentalization or relatively homogenous fluorophore distribution in the cytosol. CONCLUSION: Hamster and human resting and activated platelets can be fluorescently labeled by the lipophobicfluorescein derivatives 5,6-carboxyfluorescein and calcein as a result of fluorophore avidity to the platelet glycocalyx and sequestration by the cells. Consequently, platelets could be quantified by flow cytometry and visualized by confocal microscopy.status: publishe