Screen Quest™ Rhod-4 No Wash Calcium Assay Kit

Protocol summary

1. Prepare cells in growth medium with 1-5% FBS
2. Add Rhod-4 NW dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

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

1. Rhod-4 NW stock solution:
Add 200 µL of DMSO into the vial of Rhod-4 NW (Component A) and mix well. Protect from light. Note: 20 µL of Rhod-4 NW stock solution is enough for one plate.  Note: Unused Rhod-4 NW 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):
a) For Cat. # 36333 (1 plate kit) and # 36334 (10 plates kit) , make 1X assay buffer by adding 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B), and mix them well.
b) For Cat. # 36335 (100 plates kit), make 1X assay buffer by adding 90 mL of HHBS (Not included) into 10X Pluronic® F127 Plus (10 mL, Component B), and mix them well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 1X assay buffer at < -20 ^oC. Protect from light and avoid repeated freeze-thaw cycles.

Rhod-4 NW dye-loading solution:
Add 20 µL of Rhod-4 NW stock solution into 10 mL of 1X assay buffer, 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 Rhod-4 NW dye-loading solution into the cell plate.
   
   
2. 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 ^oC, 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.
    
3. Prepare and add 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.