Intracellular calcium concentration is known to play a significant role in both physiological and pathophysiological conditions. Recently, much attention has been given to the determination of free intracellular calcium concentration using flow cytometry. Newly developed fluorescent probes, such as Indo-I which exhibits a large spectral emission shift when it chelates Ca++ ions, have been instrumental in the development of this application. However, because calibration of the flow cytometer response with respect to the ratio of the FL1 (405-nm peak) and the FL2 (485-nm peak) is extremely difficult and time consuming, the measurement of cytosolic Ca++ by flow cytometry has been limited.This project focuses on the development of an Indo-1 Microbead Standards Kit consisting of Indo1 coated microbeads suspended in a series of solutions containing predetermined concentrations of free Call. Using this kit, the ratio of the FL1 and FL2 emission fluorescence of Indo-i microbeads can be directly correlated with free calcium concentration. Such a kit will not only make calibration of flow cytometers more convenient, but will also help to normalize data over time and among laboratories. A more accurate determination of cytosolic calcium concentration will undoubtedly improve understanding of the biological relevance of alterations in intracellular calcium concentration.
Anticipated Results:There are approximately 5,000 flow cytometers worldwide with a growth rate of 10 to 15 percent per year. If it is estimated that 20 percent of these instruments will be utilized for Ca++ determinations, then the sale of one Indo-i Microbead Standards Kit per instrument per year at $500/kit could generate a minimum of $500,000 per year.National Institute Of General Medical Sciences (NIGMS)
