Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with CCL25 in CHO-K1/Gα15/CCR9 cells. d2 acceptor fluorophore-labeled cAMP (Cat. No. 62AM4PEC; Revvity) and intracellular cAMP in CHO-K1/Gα15/CCR9 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 CCL25 on CHO-K1/Gα15/CCR9 cells was 5.64 μg/ml.

Figure 3. FACS analysis of cell surface expression of CCR9 on CHO-K1/Gα15/CCR9 cells. The CHO-K1/Gα15/CCR9 cells (Red) and the negative control CHO-K1/Gα15 cells (Green) were probed using Alexa Fluor® 647 conjugated anti-human CCR9 (Cat. No. 358912, Biolegend).

Figure 1. CCL25-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/Gα15/CCR9 cells. The cells were loaded with Calcium-4 (Cat. No. R8142; Molecular Devices) prior to being stimulated with agonist CCL25 (Cat. No. Z02847; Genscript). The intracellular calcium change was measured by FLIPR. The relative fluorescent units (∆RFU) were plotted against the log of the cumulative doses of CCL25 (Mean ± SEM, n = 3). The EC₅₀ of CCL25 on this cell was 1.27 µg/ml.

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 ∆RFU and starts at Bottom and goes to Top with a sigmoid shape.