Screen Quest™ Live Cell cAMP Assay Service Pack

1. Prepare cells for transfection

2. Prepare Transfectamine™ 5000-DNA mixture

3. Add Transfectamine™ 5000-DNA mixture to the cell culture, incubate overnight

4. Transfer the transfected cells to a 96-well plate 24-30 hours after transfection, and incubate the culture overnight

5. Add Calbryte™ 520 NW dye-loading solution

6. Incubate at room temperature or 37 °C for 30-60 minutes

7. Monitor the fluorescence intensity at Ex/Em = 490/525 nm

Thaw the kit components at room temperature before starting the experiment.

1. Seed the cells such that they will be ~60-70% confluent at the time of transfection.

2. Replace with fresh growth medium before transfection. For example, replace with 2 mL of medium per well for 6-well plates and 6 mL of medium for 10 cm plates.

1. Add 20 µL of DMSO (Component F) to the vial of Calbryte™ 520NW (Component C) for Cat# 36382 and 36383, or 200 µL for Cat# 36384, and mix thoroughly.

   Note: 20 µL of Calbryte™ 520NW stock solution is enough for one plate. Unused Calbryte™ 520NW stock solution can be aliquoted and stored at < -20 °C for more than one month, provided the tubes are tightly sealed. Protect from light and avoid repeated freeze-thaw cycles.

1. Combine 9 mL of HHBS (Component E) with 1 mL of 10X Pluronic® F127 Plus (Component D), and mix thoroughly.

1. Add 20 µL of Calbryte™ 520 NW stock solution to 10 mL of 1X Assay Buffer, and mix well.

   Note: The working solution is stable for at least 2 hours at room temperature.

1. Add 15 µL of ddH₂O to the vial of Ga16 DNA (Component A), mix well to have the final concentration of 1 µg/µL.

   Note: Skip this step for Cat# 36384. Component A is already provided in solution form and is ready to use.

2. Mix 3 µg of DNA [for example, 1.5 µg of Ga16 DNA (Component A) and 1.5 µg DNA of the GPCR that you are interested] with 200 µL of serum-free medium.

3. Add 9 µL of Transfectamine™ 5000 (Component B) to the mixture from Step 1.

4. Mix well and incubate at room temperature for 20 minutes.

   Note: The ratio of Transfectamine™ 5000 and DNA need to be optimized for different  cell lines. In general, the ratio for Transfectamine™ 5000 Transfection Reagent (µL) to DNA (µg) should be 3-5 µL : 1 µg.

Table 1. Sample  protocols for a 6-well plate and a 10 cm plate

1. Add Transfectamine™ 5000 -DNA mixture to the culture plate and incubate overnight.

   Note: The recombinant protein can start to be detected as early as 16 hours after transfection. The maximal expression level may be observed 72~96 hours after transfection.

2. Transfer the transfected cells to a 96-well plate 24-30 hours post transfection and incubate overnight.

   • For adherent cells: Plate cells overnight in the growth medium at 40,000 to 80,000 cells/well/100 µL for a 96-well plate.
   • For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in cell growth medium or HHBS at 125,000 to 250,000 cells/well/100 µL for a 96-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments.

   Note: Each cell line should be evaluated on the individual basis to determine the optimal cell density for the intracellular calcium mobilization.

1. Add 100 µL/well (96-well plate) of Calbryte™ 520NW working 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 15-30 minutes.

   Note: If the assay requires 37 °C, 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 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 = 490/525 nm.