Screen Quest™ Luminometric Calcium Assay Kit

Protocol summary

1. Prepare cells by removing growth medium
2. Add Coelenterazine-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
3. Incubate at room temperature for 3-4 hours
4. Monitor aequorin luminescence intensity

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

1. Coelenterazine analog:
Add 250 µL of 100% ETOH (Component B) into the vial of Coelenterazine analog (Component A), and mix them well. Note: 25 µL of reconstituted coelenterazine analog is enough for one plate. Unused coelenterazine analog stock solution can be 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):
a) For Cat. # 36305 (10 plates kit), ready to use 1X Assay Buffer (Component C).
b)For Cat. # 36306 (100 plates kit), make 1X assay buffer by diluting 10 mL of 10X Assay Buffer (Component C) into 90 mL of HHBS buffer (not included in the kit), and mix them well. Note: 10 mL of 1X assay buffer is enough for one plate. Store unused 1X assay buffer at 4 ^oC.

Coelenterazine-loading solution:
Add 25 µL of ETOH reconstituted coelenterazine analog (Prepartion Of Stock Solutions) into 10 mL of 1X assay buffer (Preparation Of Stock Solutions), and mix them well. Note: This working solution is stable for at least 2 hours at room temperature, protected from light.

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 plates. Note: It is important to remove the growth medium in order to minimize compound interference with serum or culture media. Note: Alternatively, grow the cells in growth medium with 0.5-1% FBS to avoid medium removal step. In this case, 2X Coelenterazine-loading solution in 1X buffer is needed.
   
   
2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) Coelenterazine-loading solution into the cell plates.
   
   
3. Incubate the Coelenterazine-loading plates at room temperature for 3-4 hours, protected from light.
   
   
4. Prepare the compound plates with HHBS or the desired buffer.
   
   
5. Monitor the aequorin luminescence intensity by using the photon detection system that has an enclosed chamber containing a photomultiplier. The instrument must completely exclude outside light.