Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus)

Abstract

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+, using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole‐blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+‐sensitive dye Fluo‐4AM and a clone of the platelet‐specific antibody CD41‐PE that recognizes dolphin platelets. This no‐wash, no‐lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal‐transduction responses to platelet agonists under near‐physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.

Flow Cytometry of Oxygen and Oxygen-Related Cellular Stress

“…Including time as an operative parameter in FCM has led to the concept of realtime flow cytometry (RT-FCM), an experimental strategy in which the changes in cellular parameters within heterogeneous cell populations are followed in a kinetic fashion [47,62]. In RT-FCM, the biological process of interest progress as the sample is run in the flow cytometer and single cells are analyzed in sequence.…”

“…RT-FCM allows to monitor functional changes in high numbers of single cells with a theoretical time-resolution of less than one second, and for time windows ranging from few seconds to several minutes. This type of kinetic analysis is specially relevant for following very fast or transient dynamic processes, as those typical of signal transduction [62].…”

“…Such histograms are clinically used to diagnose CGD or other oxidative-burst related genetic defects [58]. Panel B exemplifies graphically the performance of a flow cytometry kinetic analysis (RT-FCM or In Fluxo analysis [62]) of the ROS generation in Jurkat cells treated with exogenous H 2 O 2 or with the mitochondrial uncoupler FCCP [53]. Graph B1 shows the identification and gating of live (blue) and dead (orange) cells using FS signal and the viability stain DAPI [53] and graph B2 displays the gate used for single-cell selection in a FS Peak vs. FS INT dotplot.…”

“…Upon oxidation of DHRH123 to the fluorescent rhodamine 123, one of the two equivalent amino groups tautomerizes into an imino group, trapping rhodamine 123 within mitochondria [118]. DHRH123 is oxidized by H 2 O 2 in the presence of peroxidases, but it can also be oxidized by other reactive oxidants, as ONOO, Fe 2+ , Fe 3+ in the presence of ascorbate or EDTA, cytochrome c, or HOCl [62,81]. DHRH123 is not directly oxidized by H 2 O 2 alone, O 2…”