Cell Meter™ Beta-Arrestin Translocation GPCR Signaling Kit

1. Prepare cells for transfection
2. Prepare Transfectamine™ 5000-DNA mixture
3. Add Transfectamine™ 5000-DNA mixture to cell culture, and incubate overnight
4. Transfer the transfected cells to a 96-well plate 24-30 hours after transfection, and incubate the culture overnight
5. Analyze translocation induced by GPCR activation under a fluorescence microscope

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

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

2. Replace with fresh growth medium before transfection.

   Note: For example, replace with 2 mL of medium per well for 6-well plates and 6 mL of medium for 10 cm plates.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

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

1. Mix 3 µg of DNA [for example, 1.5 µg of Beta-arrestin-GFP DNA stock solution and 1.5 µg DNA of the GPCR that you are interested] with 200 µL of serum-free medium.
2. Add 9 µL of Transfectamine™ 5000 (Component B) to the mixture.

3. 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, in our testings, the ratio for Transfectamine™ 5000 Transfection Reagent (µL) to DNA (µg) Ratio should be 3-5 µL : 1 µg.

Table 1. Sample rprotocols 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 after transfection and incubate overnight.
3. Monitor the beta-arrestin translocation induced by the receptor activation under a fluorescence microscope with the FITC filter (Ex/Em = 488/530 nm).