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HEK293 Calcium Flux Assay

y buffer to the cells for a final cell density of 2.3 x 106 cells per mL and again triturate vigorously.

III. Assay Development

Kinetic Response

The LabChip 3000 system is designed such that calcium response for each cell is measured at a fixed incubation time. The cell velocity and the length of the incubation channel determine the incubation time. Cell velocity is a function of the pressure applied to the chip, the chip temperature and the solution viscosity; while incubation channel length can be varied by selecting detection points at three locations (or zones) along the cell path. Typically pressures of -1.2, -2 and -3 psi are sufficient to gather statistics on optimum incubation time. In this experiment, carbachol was sipped up onto the chip and contacted with cells. The ratio of fluorescence for each cell at 530 nm (Fluo-4) and 685 nm (Fura Red) was measured and plotted as a function of incubation time. From Figure 1, 30 s was determined to be the optimum incubation time for HEK293 cells stimulated by carbachol. Table 1 gives the assay conditions for the HEK293 agonist assay.

Assay Stability

Once reaction conditions were determined, the stability of the assay over the anticipated run time was assessed. Stability of the assay was assessed by examining the cell count rate and the cell viability (e.g. EC50) over time. Figure 2 shows that cell counts are > 80 cells per well after 4 hr. Figure 3 shows the stability of the cell response over a typical run time of 4 hr.

IV. Assay Validation

Typical validation criteria include data quality (Z') assessment, assay stability over multiple runs, and cell usage.

Data Quality Assessment

Figure 4 shows the calculated Z' values for the agonist assay. Ass
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