A calcium flux assay has been developed for the LabChip 3000 microfluidic system for use in drug screening against GPCR targets. The miniaturized platform results in low cellular and reagent consumption, high data quality and a high degree of automation.
The LabChip microfluidic assay accesses test compounds from a microplate and mixes the samples with suspended cells continuously flowing through the chip. Flow through the microchannel network results from a vacuum applied to the waste wells of the chip. The system measures the fluorescence properties of individual cells flowing past a fluorescence detector. The LabChip system measures as few as 50 cells and uses less than 50 nL of sample and 15 nL of agonist per measurement.
The steps involved in developing and validating an assay to conduct screens for GPCR agonists and antagonists will be discussed here. CHO-m1 is used as a model cell line.
CHO-m1 Assay Summary
Typical cell usage as low as 600-1000 cells/well
Z' = 0.86 for Agonist, Z' = 0.72 for Antagonist
Unattended run time of between 4 and 8 hr
Throughput of up to 8,000 samples in 8 hr
The general steps followed in the development of a calcium flux assay are outlined in Figure 1. A preliminary choice of cell line, growth conditions, and cell assay buffer is made (Step 1). A protocol for labeling, washing and suspending cells is then developed (Step 2), after which confirmation is made that the labeled cells can be detected (Step 3). Once feasibility has been established, the kinetics and magnitude of response to agonist exposure is measured (step 4) and, in the case of an antagonist assay, the concentration of agonist to be placed on-chip is determined (Step 5).
The dilution factor used to calculate the appropriate microplate an