Cytosolic free Ca2+ concentration ([Ca2+] cyt) has been measured in various cells by using a new fluorescent calcium indicator, quin 2. In human platelets, some reports have been published on [Ca2+] cyt associated with activation by various agonists. But little has been reported concerning about the method of measuring [Ca2+] cyt in platelets.
We investigated the method for calibration of quin 2 fluorescence, concentration of quin 2/AM and incubation time after preparation of Ca2+ concentration in the medium ([Ca2+] med). Resting [Ca2+] cyt levels were 121.3±12.9 nM in human platelets by calibration described by Wollheim. Platelet aggregation and release were absolutely inhibited by adding of 50 μM quin 2/AM and over. Initial stage of platelet activation was slightly inhibited by addition of 20 μM quin 2/AM. But quin 2 concentration in platelets didn't reach to the appropriate concentration by adding 5-10 μM quin 2/AM to PRP. Since [Ca2+] cyt levels were gradually increased within 20 min. after addition of 1 mM CaCl2, it was desirable to measure [Ca2+] cyt between 20 and 40 min. after preparation of [Ca2+] med.
If preparation of cell suspension and calibration of quin 2 fluorescence was done properly, measurement of [Ca2+] cyt with quin 2 is useful for research of Ca2+ movement, in spite of limitation of functional modulation by quin 2.
