Figure 1. Glucagon-induced concentration-dependent stimulation of intracellular calcium mobilization in HEK293/GCGR/Gα15 cells. The cells were loaded with Calcium-4 prior to being stimulated with agonist glucagon. The intracellular calcium change was normalized and measured by FLIPR. The relative fluorescent units (RFU) were normalized and plotted against the log of the cumulative doses of glucagon (Mean ± SEM, n = 3). The EC₅₀ of glucagon on this cell was 37.92 nM.

Notes:
EC₅₀ value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom) / (1+10^((LogEC₅₀-X)*Hill Slope))
X is the logarithm of concentration. Y is the response
Y is % stimulation of RFU and starts at Bottom and goes to Top with a sigmoid shape.

Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with glucagon in HEK293/GCGR/Gα15 cells. d2 acceptor fluorophore -labeled cAMP (Cat. No. 62AM4PEC; Revvity) and intracellular cAMP in HEK293/GCGR/Gα15 cells competitively bind with Europium Cryptate-labeled anti-cAMP monoclonal antibody. The FRET signal decreases as the intracellular cAMP concentration rises and was measured by plate reader (Pherastar, BMG). The EC₅₀ of glucagon on HEK293/GCGR/Gα15 cells was 5.01 nM.

Figure 3. Dose dependent stimulation of intracellular IP-One accumulation upon treatment with glucagon in HEK293/GCGR/Gα15 cells. d2 acceptor fluorophore-labeled IP-One (Cat. No. 62IPAPEB; Revvity) and intracellular IP-One in HEK293/GCGR/Gα15 cells competitively bind with Europium Cryptate-labeled anti-IP-One antibody. The FRET signal decreases as the intracellular IP-One concentration rises and was measured by plate reader (Pherastar, BMG). The EC₅₀ of glucagon on HEK293/GCGR/Gα15 cells was 0.60 μM.