During the last years flow cytometry has been developed into an important diagnostic tool with a wide range of applications. Whereas the differentiation of leukocytes is most often encountered, flow cytometry also renders itself especially useful in the study of platelets. Commonly, surface markers are of key interest, but the fact that platelets can be conveniently activated in vitro offers the opportunity to investigate intracellular signal transduction processes, which lead to the release of calcium into the cytosol. And it is this rise of intracellular calcium that flow cytometry is able to detect in real time through the use of suitable calcium-sensitive markers.
It is the aim of this book to put together an overview of this technique and therefore to provide a sound starting ground for its application to clinical and experimental investigations and also for its further development.
Table of Contents
Introduction
The Role of Calcium in Intracellular Signal Transduction
Sources of Calcium
Signal Transduction
Activation of Platelets
Adhesion
Shape Change
Aggregation
Secretion
Procoagulant Activity
Soluble Platelet Agonists
Inhibition of Platelets by cAMP
Flow Cytometry
Principle
Parameters
Monoclonal Antibodies
Isotype Control
Compensation
Monoclonal Antibodies Used for the Flow Cytometric Characterization of Platelets
Intracellular Probes
Presentation and Analysis of Data
Dotplots and Identification of Cells
Further Analysis
Regions and Gates
The Use of Fluo-3 as Intracellular Calcium Indicator
Brief Introduction
Fluo-3
Application
Flow Cytometric Analysis
Conclusion
Objectives and Research Scope
This work aims to provide a comprehensive overview of using flow cytometry to monitor intracellular calcium dynamics in platelets. It explores the physiological role of calcium as a secondary messenger during platelet activation and demonstrates how flow cytometric techniques, specifically utilizing the indicator Fluo-3, can measure these signals in real time for clinical and experimental research.
- The physiological significance of calcium in platelet signal transduction.
- Technical principles of flow cytometry for cellular analysis.
- Methodologies for characterizing platelet surface markers and activation states.
- Practical protocols for measuring intracellular calcium mobilization using fluorescent probes.
Excerpt from the Book
Sources of Calcium
The intracellular calcium concentration of resting platelets lies in the order of 40 to 100 nmol/l. Compared to the calcium concentration in the extracellular environment (i.e. in blood plasma) of roughly 2.5 mmol/l this represents a very steep concentration gradient, which is maintained predominantly by the action of a Ca2+/Mg2+ ATPase pumping the calcium mainly into the dense tubular system and perhaps outside the cell.
Upon stimulation, the concentration increases to 2 to 10 µmol/l. Calcium is released from the main storage sites, namely the dense tubular system and the plasma membrane. The dense tubular system consists of membrane-limited tubules derived from the endoplasmatic reticulum of the megakaryocytes.
Depending on the agonist, considerable amounts of calcium can be brought to the cytosol from the extracellular fluid across the plasma membrane through specific calcium channels. Additionally, calcium is located in mitochondria and dense bodies (storage granules containing serotonin among other substances), but these sites are of little importance in the activation of the platelets.
Immediately after stimulation, the calcium concentration begins to revert to basal levels indicating that the calcium is redistributed to the storage sites, mainly the dense tubular system. This is achieved by the aforementioned calcium pump, which is activated through phosphorylation by a cAMP-dependent protein kinase.
Summary of Chapters
Introduction: Outlines the development of flow cytometry as a vital diagnostic tool and its specific application in investigating platelet intracellular signaling processes.
The Role of Calcium in Intracellular Signal Transduction: Describes how calcium acts as a key second messenger in platelet activation, detailing storage sites and mechanisms of release.
Flow Cytometry: Explains the technical principles of flow cytometry, including light scattering, fluorescence detection, and signal processing methods.
The Use of Fluo-3 as Intracellular Calcium Indicator: Evaluates historical and modern fluorescent calcium indicators, focusing on the advantages of Fluo-3 for flow cytometric analysis.
Application: Details the practical laboratory procedures for preparing and analyzing Fluo-3 loaded platelets.
Conclusion: Summarizes the integration of flow cytometry as an effective method to evaluate platelet activation states by measuring cytosolic free calcium.
Keywords
Flow Cytometry, Platelets, Intracellular Calcium, Fluo-3, Signal Transduction, Haemostasis, Fluorescence, Activation, Agonists, Thrombin, TRAP-6, Cytosol, Cell Signaling, Diagnostic Tool, Spectral Compensation
Frequently Asked Questions
What is the core subject of this publication?
The publication focuses on the application of flow cytometry to measure and monitor intracellular calcium levels in human platelets during activation.
What are the primary thematic areas covered?
The main themes include the physiology of platelet activation, the role of calcium signaling, technical aspects of flow cytometric systems, and the practical usage of fluorescent indicators like Fluo-3.
What is the primary goal of the described methodology?
The goal is to provide a robust, real-time investigative method for observing how platelets respond to agonists through calcium mobilization.
Which scientific method is utilized for the investigations?
The primary method is flow cytometry, supported by the use of cell-permeant fluorescent calcium probes to detect physiological changes.
What specific topics are explored in the main body?
The main body covers signal transduction pathways, the role of various platelet agonists, the optics of flow cytometers, and step-by-step laboratory protocols for sample preparation and analysis.
Which keywords best characterize this work?
Key terms include flow cytometry, intracellular calcium, Fluo-3, platelet activation, and haemostasis.
Why is the dense tubular system important in this context?
It serves as the primary intracellular storage site for calcium in resting platelets, playing a critical role in the maintenance of the steep concentration gradient required for activation.
How does the author describe the function of the Fluo-3 indicator?
Fluo-3 is an acetoxymethyl ester dye that permeates cell membranes; once inside, it is cleaved by esterases and trapped, exhibiting a significant increase in fluorescence upon binding to cytosolic free calcium.
- Arbeit zitieren
- Thomas Gamsjäger (Autor:in), 2012, Flow Cytometry of Intracellular Calcium in Platelets, München, GRIN Verlag, https://www.hausarbeiten.de/document/190163
