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Calcium Flux Assays using the LabChip 3000

rated and the pellet was loosened by flicking prior to resuspension in 3 mL of cell assay buffer at 3-5 x 106 cells/mL (note cell assay buffer may require optimization, but an initial buffer composition given in Section IV should suffice for early phase feasibility).

3. Confirm Cell Detection
Typically, once cells have been loaded with dye and suspended in solution, the cells are added to the cell wells on the microfluidic chip. The cells are then carried to the detection point in the chip by applying a vacuum to the waste wells. Cell fluorescence excited by a 488 nm laser is detected at both 530 (Fluo-4) and 685 (Fura Red) nm. A spike or peak in fluorescence is observed as each cell passes the detector. Robust signals display peak heights greater than ten times the baseline noise.

4. Determine Kinetics of Response
The LabChip 3000 calcium flux chip is designed such that calcium response for each cell is measured at a fixed incubation time. The incubation time is determined by the cell velocity and the length of the incubation channel. Cell velocity is a function of 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. Table 1 lists the dependence of incubation time on pressure and detection zone. Typically pressures of -1.2, -2 and -3 psi are sufficient to gather statistics on optimum incubation time. In this experiment, a candidate agonist is sipped up onto the chip and contacted with cells. The ratio of fluorescence emission for each cell at 530 and 685 nm is measured and plotted as a function of incubation time. From this experiment 15 s (pressure = -2 psi at Zone 1) was determined to be the optimum incubation time for CHO-m1 cells stimulated by carbachol, since detecting at a point after the pe
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