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Calcium Flux Assays using the LabChip 3000

and washed to remove unincorporated dye. It is not critical to wash cells extensively as the LabChip assay is relatively insensitive to background fluorescence. Suspensions of adherent cell lines can be formed by a variety of methods such as trypsinizing or EDTA treatment. After lifting and washing, cells are suspended in cell assay buffer prior to loading on the microfluidic chip.

Cell Labeling
Media was aspirated from the T75 flask and cells were washed once with 10 mL HBSS with 20 mM HEPES. The HBSS/HEPES solution was aspirated and 6 mL dye loading solution was added to the flask (See Section IV). The flask was covered with aluminum foil and incubated at room temperature for 45 min. After the dye loading solution was aspirated, the cells were washed once with 10 mL of PBS without Ca2+ or Mg2+ and then with 5 mL of EDTA. The EDTA was aspirated and 2 mL of Trypsin-EDTA solution was added. After ~ 5 minutes, it was confirmed that the cells were suspended and 1 mL of Trypsin inhibitor solution was added to stop the reaction.

Suspending Cells
9 mL of cell preparation buffer (See Section IV) was added to the flask and mixed vigorously by pipette. The contents of the flask were then transferred to a 15mL centrifuge tube and centrifuged for 3 min at 200 g. The supernatant was aspirated and the pellet was loosened by flicking. 1 mL of cell preparation buffer was added and pipetted vigorously. An additional 2 mL of cell preparation buffer was added and pipetted vigorously (note: care was taken to avoid introduction of air bubbles; if introduced these can be aspirated from liquid surface). The cell suspension was incubated at room temperature for 5 minutes and an additional 4 mL of cell preparation buffer was then added and gently mixed by pipette. Cell counts were performed and the suspension was then centrifuged for 3 min at 200 x g. The supernatant was aspi
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