HTRF WT Beta-arrestin plasmid transfection principle

Stable CHO or HEK293 cells expressing GPCR are plated at 80,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2.

The medium is then removed, and 50µL of a mixture of lipofectamin + DNA in optiMEM without SVF  are added to the well. It is strongly advised to test 3 different amounts of beta-arrestin 2 plasmid to ensure identification of the best performances of the Beta-arrestin2 recruitment kit, e.g.  5, 10, & 20 ng of beta-arrestin 2 plasmid.

After 6 h of incubation at 37°C, 5% CO2, 100µL of medium with SVF are added to the well.

After 24h at 37°C, 5% CO2, the cells are ready to run various assays.

We recommend working with two different plates for the transfection process. The first plate is used to run the Beta-arrestin 2 recruitment assay on the cells treated with increasing concentrations of compounds (assessment of the assay window and EC50 for the different plasmid quantities). The second plate is used to run the HTRF Total Beta-arrestin 2 and HTRF Total AP2 assays, and assess beta-arrestin 2 and AP2 expression levels post transfection.

Performance of Beta-arrestin 2 recruitment assay as a function of the amount of WT Beta-arrestin 2 plasmid transfected into NK2R-expressing CHO cells

Stable CHO cells expressing human NK2 receptor were plated at 80,000 cells/well in 2 different 96-well plates, and incubated for 24h at 37°C, 5% CO2.

Lipofectamin transfection with 0, 5, 10, & 20 ng of beta-arrestin 2 plasmid was then performed as previously described.

The medium in the first plate was then removed, and cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. Serial dilutions of the cell lysate were performed using supplemented lysis buffer #1 (1x), and 16µL of pure sample and of each dilution were transferred into a 384-well small volume microplate before the addition of 4 µL of the HTRF Total Beta-arrestin 2 detection reagents. The HTRF signal was recorded after 3h incubation at room temperature.

In the second plate, the cells were treated with increasing concentrations of Neurokinin A diluted in Stimulation buffer 4 for 30 minutes at room temperature. The medium was then removed, and the cells were stabilized with 30 µL of Stabilization buffer 1 for 15 minutes at room temperature, then washed 3-times with 100µl of Wash buffer 1. Finally, 100µl of the B-arr2 recruitment detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature (assay protocol option 1 and 2).

The beta-arrestin 2 detection shows a signal increase with transfected Beta-arrestin 2 DNA quantity. Calculated Delta F are between the recommended values i.e. 1500 and 4000%, allowing the best assay window (S/B) in the Beta-arrestin 2 recuitment assay. Moreover, the EC50 of NeurokininA is in agreement with literature and the S/B fully improved with overexpression due to Beta-arrestin 2 plasmid.

Delta F	NT	5 ng B-arr2	10 ng B-arr2	20 ng B-arr2
PURE	267%	1608%	2660%	3819%
1/2	199%	871%	1328%	2992%

 

 

Beta-Arrestin 2 recruitment assays on WT B-arrestin 2 plasmid transfected human GLP1R-expressing HEK and CHO cells

Stable CHO or HEK293 cells expressing human GLP1 receptor were plated at 80,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2.

Lipofectamin transfection with 5, 10, & 20 ng of beta-arrestin 2 plasmid was then performed as previously described.

After treatment for 30 minutes at room temperature with increasing concentrations of Exendin-4 diluted in Stimulation buffer 4, the medium was removed. The cells were stabilized with 30 µL of Stabilization buffer 1 for 15 minutes at room temperature, then washed 3 times with 100µl of Wash buffer 1. Finally, 100µl of the B-arr2 recruitment detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature (assay protocol option 1 and 2).

The results are presented for the beta-arrestin 2 DNA amount that gives the best signal to background (S/B) for each cell line i.e. 20 ng for HEK293 GLP1 cell line, and 5 ng for CHO GLP1 cell line. In both cases, the transfection enables the S/B to be increased by roughly 30%.

Beta-arrestin 2 recruitment assays on WT B-arrestin 2 plasmid transfected MOR-expressing CHO cells

Stable CHO expressing human MOR receptor were plated at 80,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2.

Lipofectamin transfection with 5, 10, & 20 ng of beta-arrestin 2 plasmid was then performed as previously described.

After treatment for 30 minutes at room temperature with increasing concentrations of DAMGO diluted in Stimulation buffer 4, the medium was removed. The cells were stabilized with 30µL of Stabilization buffer 1 for 15 minutes at room temperature, then washed 3 times with 100µl of Wash buffer 1. Finally, 100µl of the B-arr2 recruitment detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature (assay protocol option 1 and 2).

The results are presented for the beta-arrestin 2 DNA amount that gives the best signal to background (S/B) for each cell line, i.e. 5 ng in this experiment. In both cases, the transfection enables the S/B to be increased by 30-40%.

 

Beta-arrestin 2 recruitment assay	NT	5 ng B-arr2	Percentage S/B increase vs NT
S/B protocol 2	1.3	2	43%
EC50 (nM)	85	110