A method for loading equine platelets with the fluorescent calcium indicator Fura-2: ADP induces a rise in the cytosolic free calcium ion concentration.

The research attempts to devise a method for charging horse platelets with a fluorescent calcium indicator, Fura-2. A key finding in the study indicates that the removal of plasma from the extracellular medium plays a pivotal role in successful cell charging. Additionally, the method is put to test by assessing the human platelets’ loading.

Platelets and Fura-2

• The experiment involves the use of equine platelets in platelet-rich plasma, which are then incubated with a fluorescent marker dye, Fura-2-AM (Fura-2-acetoxymethyl ester).
• The researchers then assessed the cell loading degree with the dye and the ester’s hydrolysis extent using quantitative fluorimetry and chromatography techniques.
• However, under these circumstances, the cells loaded poorly with Fura-2 to a concentration of 4 microM.

Validation and Adaptation of Technique

• The technique was validated by demonstrating the successful loading of human platelets with Fura-2, up to a concentration of 250-300 microM, utilizing the same method being tested.
• This helped validate the scientific process while also helping to determine its effectiveness and adaptability to different use cases, in this case, human platelets.

Importance of Plasma Removal

• The extraction of plasma from the extracellular environment appeared to be important for the successful dye loading of the cells.
• When cells were washed and then resuspended in a physiological salt solution, they loaded adequately with Fura-2 to a concentration of 190 microM. This points to the importance of plasma removal in this method.

Assessment of Calcium Levels Post-Stimulation

• The cells that were loaded in such a way demonstrated a resting calcium concentration of 129 nM.
• When triggered by the pro-aggregatory agonist adenosine 5′-diphosphate (ADP), there was a concentration-dependent increase in calcium levels. They rose to a temporary maximum of 350 nM. This demonstrates the cells’ response to ADP stimulation.