Fluorescent labeling of endogenous platelets for intravital microscopy: Effects on platelet function.

Monitoring endogenous platelets during intravital microscopy often involves two approaches: fluorescently labeled antibodies or genetic models of platelet-specific fluorescent protein expression. Due to limited data available on platelet functional changes induced by these methods, we compared functional effects of these methods on platelets. Platelet aggregation to collagen and thrombin, and collagen matrix-mediated platelet adhesion/aggregation under flow were tested. We assessed platelets from mice expressing EYFP on platelets (Cre(+)), littermate controls (Cre(-)), C57BL/6 mice, and platelets from vehicle control and x-488 treatment. We utilized intravital microscopy to monitor platelets in vivo using Cre(+) mice and x-488 treatment. Both genetic and antibody-based approaches yielded substantial platelet-specific fluorescence. Platelets from Cre(+) and Cre(-) mice behaved similarly in aggregation and adhesion/aggregation under flow. However, they exhibited significantly enhanced aggregation and higher adhesion/aggregation as compared to platelets from C57BL/6 mice. Compared to vehicle control, x-488 platelet labeling did not induce significant functional changes in vitro. Both methods of platelet labeling provided satisfactory platelet detectability in vivo. x-488 antibody labeling of platelets induced less alteration of platelet function than genetic approaches under our experimental conditions and seems more suitable for monitoring of endogenous platelets.