Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*

Protocol summary

1. Prepare cells
2. Remove the growth medium
3. Add Fluo-8 NW dye working solution
4. Incubate at RT for 1 hour
5. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Do not add additional probenecid. It is recommended to incubate the dye working solution no longer than 2 hours.
Thaw all components to room temperature before beginning protocol.

1. Fluo-8 NW stock solution:
For Cat No. 36307, add 10 µL of DMSO into Fluo-8 NW (Component A), and mix them well.
For Cat No. 36308 and 36309, add 100 µL of DMSO into Fluo-8 NW (Component A), and mix them well. Note: 10 µL of Fluo-8 NW stock solution is enough for 1 plate.

2. Assay Buffer stock solution (1X):
For Cat No. 36307 and 36308, add 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B) and mix well.
For Cat No. 36309, add the whole bottle of 10X Pluronic® F127 Plus (10 mL, Component B) into 90 mL of HHBS buffer (not included in kit) and mix well. Note: 10 mL of 1X Assay Buffer is enough for one plate.

Add 10 µL of Fluo-8 NW DMSO stock solution into 10 mL of 1X assay buffer and mix well. This working solution is stable for at least 2 hours at room temperature.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Remove the growth medium from the cell plate. Note: It is important to remove the growth medium in order to minimize background fluorescence and compound interference with serum or culture media. Note: Alternatively, grow the cells in growth medium with 0.5% - to 1% FBS to avoid medium removal step. In this case, 2X dye loading solution in HHBS buffer is needed. [We offer 2 separate no wash calcium assay kits (Cat No. 36315 and Cat No. 36316) for those who use 0.5% to 1% FBS in growth medium to avoid the medium removal step].
   
   
2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-8 NW dye working solution into the cell plate.
   
   
3. Incubate the dye-loading plate in a cell incubator for 30 minutes, and then incubate the plate at room temperature for another 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation. Note: If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1 - 2 hours (It is recommended that the incubation time be no longer than 2 hours.)
   
   
4. Prepare the compound plates with HHBS or your desired buffer.
   
   
5. Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 490/525 nm. Note: It is important to run the signal test before your experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum intensity counts. For example, the maximum fluorescence intensity count for FLIPR-384 is 65,000, so the instrument setting should be adjusted to have its signal test intensity around 7,000 to 10,000.