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Stock cultures of L929 cells (mouse fibroblasts) are maintained at standard culture conditions until the cells reach the desired confluency.
When cells have reached the appropriate confluency, the cells are trypsinized, counted, and seeded into Transwell™ capsule cups. The seeded cups are incubated at standard culture conditions for at least 16 hours until use in the assay.
Chamber Sensor Preparation
The Transwell™ is placed into the cytosensor chamber after at least 16 hours of incubation.
Stabilization and Baseline Rate
The chambers are transferred to the Cytosensor Microphysiometer. The L929 cells are ready for stabilization.
During the stabilization period, rates of cellular metabolism are displayed every minute for at least one hour.
At the end of the hour, at least 5 control rates are determined and averaged for each chamber. This average value is used as the baseline for the individual chamber.
Test Material Dilution
A serial dilution is prepared for each test article, with each dilution series covering a range of at least 7 concentrations.
Test materials must be aqueous soluble to be tested in this assay system.
Dosing & Rate Data
After baseline rates are determined for all of the active chambers, a 20 minute dosing cycle begins.
In this cycle, each test material concentration (dose) is added to the chambers for a 13.5 minute period. The cycle continues with a 6-minute rinse with a Low-Buffered DMEM to remove the dose. After the rinse cycle is complete, an acidification (metabolic) rate is determined within 30 seconds.