Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit

Protocol summary

1. Prepare cells in growth medium
2. Add Calbryte™ 590 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
3. Incubate at room temperature or 37°C for 45-60 minutes
4. Monitor fluorescence at Ex/Em = 540/590 nm

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

1. Calbryte™ 590 AM stock solution:
Add 20 µL (Cat. # 36200) or 200 µL (Cat. # 36201 and # 36202) of DMSO into the vial of Calbryte™ 590 AM (Component A) and mix them well. Note: 20 µL of Calbryte™ 590 AM stock solution is enough for one plate. Unused Calbryte™ 590 AM stock solution can be aliquoted and stored at < -20 ^oC for more than one month if the tubes are sealed tightly. Note: Protect from light and avoid repeated freeze-thaw cycles.

2. Assay buffer (1X):
Mix 9 mL of HHBS (Component C, not included in the kit Cat. # 36202) with 1 mL of 10X Pluronic^® F127 Plus (10X) (Component B) and mix them well.

Calbryte™ 590 AM dye-loading solution:
Add 20 µL of Calbryte™ 590 AM stock solution into 10 mL of Assay Buffer (1X) and mix them well. Note: This working solution is stable for at least 2 hours at room temperature. Note: 10 mL dye-loading solution is enough for one 96-wells plate.

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 Calbryte™ 590 AM dye-loading solution into the cell plate.
   
   
2. Incubate the dye-loading plate in a cell incubator for 60 minutes, and then incubate the plate at room temperature for another 15 - 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation. If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1 hour (It is recommended that the incubation time be no longer than 2 hours.)
   
   
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 = 540/590 nm.