Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*

Protocol summary

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

Important notes
Do not add additional probenecid. Thaw 1 vial of Rhod-4 NW (Component A), 1 bottle of 10X Pluronic^® F127 Plus (Component B), and 1 bottle of HHBS (Component C) at room temperature before use.

1. Rhod-4 NW stock solution:
Add 100 µL of DMSO into the vial of Rhod-4 NW (Component A) and mix well. Protect from light. Note: 10 µL of Rhod-4 NW stock solution is enough for one plate.

2. Assay Buffer (1X):
a) For Cat. #36330 (1 plate kit) and # 36331 (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. # 36332 (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 10 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. Remove the growth medium from the cell plate. Note: It is important to remove the growth medium in order to minimize background fluorescence, and compound interference with serum or culture media. Alternatively, grow the cells in growth medium with 1 - 5% FBS to avoid medium removal step. In this case, 2X dye loading solution in HHBS buffer is needed. [We offer 2 separate no wash calcium assay kits (Cat. #36334 and Cat. #36335) for those who use 0.5 - 1% FBS in growth medium to avoid the medium removal step].
   
   
2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Rhod-4 NW dye-loading solution into the cell plates.
   
   
3. Incubate the dye-loading plate in a cell incubator for 30 minutes, then incubate the plate at room temperature for another 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 - 2 hours.
   
   
4. Prepare the compound plate with HHBS or the desired buffer.
   
   
5. Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 540/590 nm.