Preparing fluorescent antibodies or genetic markers for identifying hormone and neurotransmitter receptor activity is not only difficult but also time-consuming. This article introduces a useful alternative method for recording physiological response changes resulting from the binding of agonists to their homologous receptors (many of which are G protein coupled receptors). When agonists bind to G protein coupled receptors, they can trigger a series of rapid cascade reactions, typically leading to the instantaneous release of calcium ions from intracellular storage sites. In the past, transient changes in intracellular calcium ion concentration ([Ca2+] i) have been used in flow cytometry to identify functional receptor expression in cell subpopulations [2]. Here, we found that the new CytoFLEX analyzer's peristaltic sample delivery system is particularly suitable for agonist based calcium ion studies. The ester based green fluorescent calcium indicator Fluo-4 AM (LifeTechnologies) can be used to measure the [Ca2+] i changes observed in HEK-293 cells in response to ATP stimulation. Simply improve the pipeline of CytoFLEX analyzer to make it easier to enter the sample tube for the application of agonists; Further improvements can be made by implementing the "stop time" detection method. By using agonists as physiological criteria for our experiments, we were able to identify receptors and conclusively demonstrate their functionality. (Author: Ira Schieren1 Peter Racanelli Damian Williams3)
Unit: 1 Howard Hughes Medical Institute, Columbia University Medical Center 2 Beckman Coulter Inc., Miami, United States 3. Department of Pathology and Cell Biology, Columbia University Medical Center)