United States
GPCR drug discovery calcium assay kits
High baseline fluorescence
- Growth medium and organic anion transport contribute to high baseline fluorescence >> remove the medium before adding indicator dye to the wells. Use dye solution within 2 hours at room temperature.
Untreated cells have calcium response
- Inconsistent DMSO concentration >> insure that buffer used for the negative control wells has the same final concentration of DMSO as those in the test compounds.
Well-to-well variation observed
- Incorrect dispensing and/or experimental setup parameter used >> a fluorescence microplate reader with automatic dispensing should be used for assays requiring precise timing, such as ion channel assays. Faster addition speeds can lead to better mixing of compounds and lower signal variance across the plate. Make sure to follow the recommended experimental setup parameters provided by the instrument manufacturer before reading the plate. It is also important to run the signal test before the experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum instrument intensity counts.
Fluorescence drop upon compound addition
- Cells may become dislodged during addition >> Decrease the rate of addition or seed fewer cells per well.
