Screen Quest™ Fluo-4 No Wash Calcium Assay Kit

Protocol summary

1. Prepare cells in growth medium
2. Add Fluo-4 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
3. Incubate at 37°C for 1 hour
4. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Do not add additional probenecid. Thaw all the kit components at room temperature before use.

1. Fluo-4 AM stock solution:
Add 200 µL of DMSO into the vial of Fluo-4 AM (Component A), and mix them well. Note: 20 µL of Fluo-4 NW stock solution is enough for one plate. Unused Fluo-4 AM stock solution can be aliquoted and stored at < -20 ^oC for more than one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles.

2. Assay Buffer (1X):
Make 1X Assay Buffer by adding 1 mL of 10X Pluronic^® F127 Plus (10 mL, Component B) into 9 mL of HHBS buffer (Component C) and mix well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 10X Pluronic^® F127 Plus (Component B) at < -20 ^oC. Protect from light and avoid repeated freeze-thaw cycles.

Fluo-4 AM dye-loading solution:
Add 20 µL of Fluo-4 NW 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. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-4 AM dye-loading solution into the cell plate.
   
   
2. Incubate the dye-loading plate in a cell incubator for 1 hour, and then incubate the plate at room temperature for another 15 to 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation.
   
   
3. Prepare the compound plate with HHBS or your desired buffer.
   
   
4. Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 490/525 nm.