Generation of fluorescent antibody or genetic labels to identify hormone and neurotransmitter receptoractivity can be difficult and time consuming. A useful alternative is recording physiological changes inresponse to agonist binding its cognate receptor, many of which are G-protein coupled. When an agonist binds a G-protein coupled receptor, it triggers a quick cascade of events that often results in a transient release of calcium from intracellular stores. Alterations in transient intracellular calcium ([Ca2+]i) levels have been used previously in flow cytometry to identify functional receptor expression in cellular subpopulations [2], here we show that peristaltic sample delivery of the new CytoFLEX analyzer is particularly well suited to agonist-based calcium studies. Using the ester based, green fluorescent calcium indicator, Fluo-4 AM (Life Technologies), [Ca2+]i changes were measured in HEK-293 cells in response to ATP stimulation. Simple plumbing modifications to the CytoFLEX allowed easier access to the sample tube for agonist application; further modifications were made to implement a “stop time” technique. By using response to agonist as our physiological criteria, we have fundamentally enabled receptor identification and conclusively demonstrated its functionality.
CytoFLEX Instrument Evaluation Using Biological Specimens
Evaluation of the Xitogen flow cytometer, by independent research flow cytometry laboratories and Beckman Coulter senior research and development scientists, was performed using flow cytometers equipped with 12 parameters and 9 fluorescent detectors. Instruments were equipped with the enhanced Side Scatter channel (VSSC) option. Evaluation of biological specimens included fresh human whole blood, human leukocyte control cells, and smaller cells and particles, such as RBCs, platelets, microbes, liposomes, and vesicles.
Forgalmazott termékeink gyártói - keressen gyártó szerint a logóra kattintva
