Abstract
Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar+-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.
Highlights
Cytosolic free calcium ions [Ca2+]i function as important second-messengers in platelets [1]
In this study we describe a novel method to quantify intraplatelet [Ca2+]i dynamics by ratiometric flow cytometry using Fluo-4 and Fura Red
We provide equations to calculate [Ca2+]i levels in a 2-kd model as well as a ready-to-use protocol to quantify intraplatelet [Ca2+]i dynamics in whole blood and platelet-rich plasma (PRP) by flow cytometry
Summary
Cytosolic free calcium ions [Ca2+]i function as important second-messengers in platelets [1]. The resulting increase in cytosolic [Ca2+]i is essential for platelet degranulation via fusion of vesicles with the plasma membrane as well as rearrangement of actin filaments and crucial for platelet shape change and aggregation. Quantitative Dual-Dye Cytometric Calcium Ion Measurements in Platelets. Several fluorescent dyes have been developed to record [Ca2+]i dynamics in isolated platelets using cuvette-based methods: In contrast to the fluorescent Ca2+ indicator Quin-2 [2] that interferes with platelet function [3], Fura-2 displays a higher quantum yield and allows ratiometric measurements. The popular dye Indo-1 allows ratiometric measurements at two emission wavelengths (excitation at 335 nm, emission at 400 and 475 nm)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
