Pairing the Green Upward and Downward DAG sensors with R-GECO makes it possible to simultaneously measure DAG and Ca²⁺ signaling in single cells.

(A) The Green Upward DAG sensor response is considerably slower than the red Ca²⁺ response in response to carbachol stimulation of the M1 receptor. (B) Similar kinetics occur with the Downward DAG sensor. (C) The two sensors can be activated independently: ionomycin, which should raise intracellular Ca²⁺ without affecting DAG levels produces a change in R-GECO but not Downward DAG, while the subsequent addition of PDBu activates Downward DAG (arrows indicate stimulus artifact).