If the assay is deemed stable, then validation will continue with optimization of throughput and cell usage and confirmation of assay stability over multiple runs (Steps 8 and 9). If the assay is not stable, then optimization of buffer conditions and cell growth conditions will be required prior to validation.
III. Assay Development Steps
1. Choose Cell Line, Cell Growth and Cell Buffer Conditions
A microfluidic assay using the CHO-m1 cell line was developed. Cells were grown in Ham's F12 medium adjusted to contain 1.5 g/L sodium bicarbonate with 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM L-glutamine, 0.1 mg/mL Geneticin G418, 1 mM sodium pyruvate, 10 mM HEPES and 10% Fetal Bovine Serum.
Cells were maintained at 37 oC and 5% CO2 and subcultured in monolayers in T75 flasks using standard cell culture methodologies. Cells were split at 80-90% confluency. The day before each assay cells were seeded at 3 x 106 cells into T75 flasks and incubated overnight at 37 oC and 5% CO2.
2. Label, Suspend and Wash Cells
Cells are labeled at room temperature with two calcium sensitive dyes , Fluo4 and Fura Red, while attached to the plate to minimize cell stress due to multiple pelleting and suspension steps. The minimum concentration of dye that yields bright fluorescently-labeled cells should be used. Typically, 6 x 106 cells are loaded with 6 mL of physiological buffer containing 1 μM Fluo-4 and 1 μM Fura Red. In addition, anion transport inhibitors such as probenecid should be used for cell lines such as CHO-m1 that rapidly export Ca2+ indicators. After loading of dye, the cells are suspended