5. Determine On-Chip Agonist Concentration
For antagonist assays, the concentration of agonist to be placed on the chip is determined. The CHO-m1 assay was run with on-chip agonist concentration equal to the compound EC80.
EC80 is determined by placing increasing concentrations of agonist in the wells of a microplate and determining cell response. This response is compared to a control response such as ionomycin and a % activity is calculated. Analysis of the dose-response curve (Figure 4) yielded an EC50 of ~ 0.07 μM and an EC80 of ~ 0.6 μM. The concentration of agonist in the agonist well on the chip was then set at 6 μM to yield 0.6 μM in the reaction channel of the chip.
6. Determine Compound Plate and 100% Control Concentrations
Compound concentrations in microplates and control troughs must be chosen to account for on-chip dilution from cell sidearms and dispersion in the main channel. Sidearm dilutions for agonist and antagonist chips is ~50%. Sample dilution due to dispersion depends on assay parameters such as sample sip time, detection zone and applied pressure. For agonist and antagonist chips, typical operating conditions of -2 psi, (zone 1), and sample sip times > 5 s result in effectively no dispersion-related dilution of compound from the sipper. On-plate and 100% control compound concentrations were therefore doubled to account for on-chip dilutions.
7. Assay Stability
Once reaction conditions have been determined, the stability of the assay over the anticipated run time can be assessed. Typical run times for cell-based assays range from 4 to 8 hr. Stabili