Dataset Information

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

ABSTRACT: OBJECTIVE: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. METHODS: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. RESULTS: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. CONCLUSIONS: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.

SUBMITTER: Da Q

PROVIDER: S-EPMC6204114 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Da Qi Q Derry Paul J PJ Lam Fong W FW Rumbaut Rolando E RE

Microcirculation (New York, N.Y. : 1994) 20180530 6

<h4>Objective</h4>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.<h4>Methods</h4>Platelet aggregation to collagen and thrombin, and collagen matrix-mediated platelet adhesion/aggregation under flow were ...[more]

PMID: 29701894

Similar Datasets

Project description:Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy--the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets