IP1 accumulation assay

IP₁ concentrations in CCR5-transfected cells were measured using a competitive homogeneous time-resolved fluorescence assay after incubation with each chemokine for 3 hours. For each well of a low-volume, 384-well plate, HEK293T cells (5000 in 7 μl of DMEM) were transfected with 11 ng of CCR5 vector or 11 ng of empty vector. Where applicable, 5.5 ng of G_q, G_i2, or G_qi5 vector constructs was cotransfected. Twenty-four hours after transfection, cells were stimulated by chemokine diluted in 7 μl of 1× stimulation buffer [10 mM Hepes, 1 mM CaCl₂, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, and 50 mM LiCl (pH 7.4)] with 0.2% BSA and 50 mM LiCl (to prevent IP₁ degradation). The chemokine was incubated at 37°C for 3 hours. After incubation, cells were lysed by the addition of d2-fluorophore–labeled IP₁ analog (3 μl per well) as the fluorescence acceptor and the terbium cryptate–labeled anti-IP₁ monoclonal antibody as the fluorescence donor. Both fluorescent donor and acceptor were diluted in the kit-supplied lysis buffer. The plates were incubated overnight at 4°C, and time-resolved fluorescence signals were read using the BioTek Synergy NEO plate reader (BioTek Instruments, Winooski, VT) at 620 and 655 nm. Results were calculated as a 665-nm/620-nm signal ratio, and IP₁ concentrations were interpolated from a standard curve prepared using the supplied IP₁ calibrator. Results are shown as picomoles of IP₁ formed per well. For experiments involving PTX, cells were treated with PTX (100 ng/ml) for 16 hours at 37°C and 5% CO₂ before stimulation. For experiments involving YM-254890, 10 μM YM-254890 or equivalent DMSO vehicle was added to the cells and incubated at 37°C for 30 min.