pHSense Eu Anti-FLAG, 2 x 96 wells

pHSense Eu Anti-FLAG Assay Protocol

The assay begins by culturing cells in a 96-well plate. The pHSense Eu Anti-FLAG is then added to the cells and incubated for 1 hour at room temperature. Following this incubation, cells are stimulated with a pharmacological compound. Fluorescence is then measured either kinetically or at endpoint using an HTRF-compatible plate reader.

The pHSense Eu Anti-FLAG was tested on 2 different stable cell lines expressing various level of receptors at the membrane: Tag-Lite® GLP1R cells (HEK-GLP1R) and Tag-lite® Mu opioid cells (CHO-MOR).

The relative levels of receptor expression levels were determined using the Tag-lite SNAP-Lumi4-Tb Labeling Reagent (Part#: SSNPTBC, Revvity). Cells were seeded in a 96-well black culture-treated plate at a density of 100,000 cells per well in complete culture medium. After overnight incubation at 37°C with 5% CO₂, the supernatant was removed and 100 µL of 100 nM SNAP-Lumi4-Tb Labeling Reagent were added to the cells, followed by a 1h incubation at 37°C with 5% CO₂. Cells were washed 3 times with Tag-lite SNAP/CLIP Labeling Medium 1X (Part#: LABMED, Revvity) before the addition of 100 µL of the same medium. The 620 nm signal was recorded using an HTRF-compatible plate reader. Results showed Tag-Lite® GLP1R cells exhibit a 5-times higher expression level than Tag-lite® Mu opioid cells.

To run the pHSense Eu Anti-FLAG assay, Tag-Lite® GLP1R and Tag-lite® Mu opioid stable cell lines were seeded in a 96-well white culture-treated plate at a density of 80,000 cells per well in complete culture medium, and then incubated overnight at 37°C with 5% CO₂.

After cell supernatant removal, 40 µL of the pHSense Eu Anti-FLAG diluted in cell culture medium were added to the cells and incubated for 1 hour at room temperature. Exendin-4, a GLP1R agonist, and DAMGO, a Mu opioid agonist, were diluted in cell culture medium, and 10 µL of agonists or cell culture medium - as constitutive internalization condition - were added to the wells. After incubation at 37°C, the signal was recorded using an HTRF-compatible plate reader.

In parallel, the same assay was performed with the pHSense Eu Anti-FLAG and receptor agonists prepared in Tag-lite SNAP/CLIP Labeling Medium as physiological buffer.

As demonstrated here, the pHSense Eu Anti-FLAG allows the detection of agonist induced and constitutive internalization in cell lines with different receptor expression levels, and is compatible with Tag-lite SNAP/CLIP Labeling Medium as physiological buffer or cell culture medium.