Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit *Optimized for microplate reader*

Protocol summary

1. Prepare cells in growth medium
2. Add Fluo-8E™ 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 60 minutes
4. Add 50 µL calcium flux stimulator
5. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. Fluo-8E™ AM stock solution:
Add 10 µL of DMSO into the vial of Fluo-8E™ AM (Component A) and mix well. Note: 10 µL of Fluo-8E™ AM stock solution is enough for 50 assays (Half of the 96-well plate).  Note: Unused Fluo-8E™ 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):
Add 9 mL of HHBS (Component C) into 10X Pluronic^® F127 Plus (1 mL, Component B), and mix them well. Note: 10 mL of Assay Buffer (1X) is enough for one plate. Aliquot and store unused 1X assay buffer at < -20 °C. Protect from light and avoid repeated freeze-thaw cycles. 

Fluo-8E™ AM dye-loading solution:
Add 10 µL of Fluo-8E™ AM stock solution into 5 mL of Assay Buffer (1X), and mix them well. Note: 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-8E™ AM dye-loading solution into the cell plate. Do not remove the growth medium from the cell plate.
   
   
2. Incubate the dye-loading plate in a 5% CO₂ incubator at 37^oC for 45 - 60 minutes.
    
3. Prepare the Calcium stimulator solution (5X) with HHBS or your desired buffer.
   
   
4. Add 50 µL of the prepared stimulator and run the calcium flux assay immediately by monitoring the fluorescence intensity at Ex/Em = 490/525 nm (Cutoff=515 nm) with bottom read mode. Note: To achieve the best results, it is important to run the assay within 1 minute after the addition of the agonist. It is also important to make sure the time between the agonist addition and the beginning of the actual reading stays constant for all the samples.