QUANTITATIVE IND-1 MICROBEAD STANDARD IN FLOW CYTOMETRY

INTRACELLULAR CALCIUM CONCENTRATION IS KNOWN TO PLAY A SIGNIFICANT ROLE IN BOTH PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL CONDITIONS. RECENTLY, MUCH ATTENTION HASBEEN GIVEN TO THE DETERMINATION OF FREE INTRACELLULAR CALCIUM CONCENTRATION USING FLOW CYTOMETRY. NEWLY DEVELOPEDFLUORESCENT PROBES, SUCH AS INDO-1 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 INDO-1 COATED MICROBEADS SUSPENDED IN A SERIES OF SOLUTIONS CONTAINING PREDETERMINED CONCENTRATIONS OF FREE CA(++). USING THIS KIT, THE RATIO OF THE FL1 AND FL2 EMISSION FLUORESCENCE OF INDO-1 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 UNDERSTANDINGOF THE BIOLOGICAL RELEVANCE OF ALTERATIONS IN INTRACELLULAR CALCIUM CONCENTRATION.