Poster (download)
212
Darya V. Spiryova1, Alexander E. Moskalensky2, Alexei Vorob’ev3
1NSU, gerd.raz@yandex.ru
2NSU, a.mosk@nsu.ru
3NSU, vor@nioch.nsc.ru
Activation of blood platelets is the main process in normal hemostasis. Unfortunately, it also makes a great contribution to the development of cardiovascular diseases. In this work, we study activation dynamics using cytosolic calcium probe. To increase the accuracy of dynamic measurements, we present a method for optical activation of single platelets in suspension. It is achieved by the use of photolabile caged activation agonists. Futhermore, a mathematical model was developed that allows to detect individual calcium peaks andВ derive their dynamic parameters.
Thanks for the interesting presentation.
You have changed the name of the presentation. From the title of the theses it followed that method cytosolic calcium probe can be used to monitoring platelet activation (Is that so?). And the title of the presentation indicates that dual agonist optical stimulation of platelet results in increased and reliable activation. Since there is no conclusion in the work, it is not clear how obtained information can be applied.
Thanks for the interesting question.
This information allows us not only to increase the overall number of active cells, but also to avoid UV-cytotoxicity. As you can see in the graph (percentage of active cells), using two agonists allows us to irradiate cells for 10 ms and 50 ms and get more reproducible activation than when irradiated with 100 ms and 500 ms, which suggests that UV irradiation also plays an important role in the probability of cell activation in the sample. The results open the way to optical activation of platelets by agonists other than ADP, since epinephrine can also potentiate this process.In the future, we planned to improve the efficiency of experiments even more by optimization of the sample preparation protocol.
Please could you explain how exactly the calcium level is detected? It is not crystal clear from the presentation
From your text:…»An inverted microscope (Carl Zeiss AxioVert. A1) is used to monitor the intracellular calcium level during platelet activation…»
Thank you for the question!
The monitoring of cytosolic calcium in platelets was performed with a fluorescence microscope using a Fluo-4 AM calcium probe. AxioCam 503 Mono camera was used to record the dynamics of fluorescence in each cell at a speed of 3 frames per second. To get the dependence of fluorescence on time, a plugin TrackMate was used that allows us to track each individual cell.
Thank you for kindly reply