Application Note

Rapid Screening Method of Calcium Flux by Flow Cytometry

The unique peristaltic sample delivery system of the CytoFLEX analyzer enables optimized measurements of transient changes in intracellular calcium in cells following agonist activation. 

Generation of fluorescent antibody or genetic labels to identify hormone and neurotransmitter receptor activity can be difficult and time consuming. A useful alternative is recording physiological changes in response 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+] changes were measured in HEK293 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.

IN THIS PAPER YOU WILL LEARN

  • How to set up a flow cytometeric assay to measure changes in cellular of calcium
  • How to adapt the CytoFLEX flow cytometer to make real-time measurements for cellular assays
  • About a rapid screening method of calcium flux by flow cytometry