Figure 1. CCL3-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/Gα15/CCR1 cells. The cells were loaded with Calcium-4 (Cat. No. R8142; Molecular Devices) prior to stimulation with CCR1 agonist, CCL3 (Cat. No. Z03137, Genscript). The intracellular calcium change was measured by FLIPRTETRA. The relative fluorescent units (RFU) were recorded and plotted against the log of the cumulative doses of CCL3 (mean ± SEM, n = 3). The EC₅₀ of CCL3 on CHO-K1/Gα15/CCR1 cells was 0.63 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 along a sigmoid curve.

Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with CCL3 in CHO-K1/Gα15/CCR1 cells. d2 acceptor fluorophore-labeled cAMP (Cat. No. 62AM4PEC; Revvity) and intracellular cAMP in CHO-K1/Gα15/CCR1 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 CCL3 on CHO-K1/Gα15/CCR1 cells was 0.32 μg/ml.

Figure 3. FACS analysis of cell surface expression of human CCR1 on CHO-K1/Gα15/CCR1 cells. The CHO-K1/Gα15/CCR1 cells (green) and the negative control CHO-K1/Gα15 cells (red) were probed using Anti-human CCR1 antibody and Alexa FluorTM 488-conjugated goat anti-human IgG (H+L) (Cat. No. A11013, Invitrogen).