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GPCR-mediated signaling

 Figure 1 GPCR assays using recombinant cells. CHOK1 cells expressing the -Adrenergic 2A (CHO--2A), Histamine H1 (CHO-H1) or Dopamine D1 (CHO-D1) receptor were seeded at 12,000 cells per well on 384 well E-Plates, grown overnight, and the growth media was changed for Assay Buffer (left panels) or left in place (middle panels). After 15-60 minutes, cells were treated with the indicated concentrations of agonist and monitored for morphological responses on the xCELLigence system. The Cell Index value was normalized for each well to the time point immediately preceding agonist addition (indicated by arrow and plotted as the mean value from a minimum of four replicates (left and middle panels). Maximal (or minimal, in the case of dopamine tested in Assay Buffer) Normalized CI values were used to plot dose responsiveness as mean values with error bars representing one standard deviation (right panels).

Figure 2 GPCR assays on endogenous receptors. HeLa cells were seeded at 6,000 cells per well on 384 well E-plates, grown overnight, then treated with the indicated concentrations of agonist and monitored for morphological responses on the xCELLigence system. The Cell Index value was normalized for each well to the time point immediately preceding agonist addition (indicated by arrow) and plotted as the mean value from a minimum of four replicates (left panels). Maximal Normalized CI values were used to plot dose responsiveness as mean values with error bars representing one standard deviation (right panel).

Figure 3. GPCR Antagonist Assay CHO-K1 cells expressing the -Adrenergic 2A receptor were seeded at 12,000 cells per well on 384 well E-Plates, grown overnight, and the growth media was changed for Assay Buffer. The indicated antagonist was added, and after 1 hr incubation the -Adrenergic 2A receptor-selective agonist UK 14,304 was added at 100nM. The resulting dose-response relationship is plotted here, with mean values from four replicate wells, and error bars representing one standard deviation. ARC 239 is a selective Adrenergic α2B receptor antagonist, BRL 44408 is a selective Adrenergic α2A receptor antagonist, and Rauwolscine is a potent, general Adrenergic α2 receptor antagonist.

Advantages

• Highly sensitive and robust
• Modulate GPCR activity in both recombinant cell lines as well as with endogenously expressed receptors, in both agonist and antagonist modes
• Monitor responses on a wide variety of GPCR families including all coupling classes.