pHSense Eu SNAP Labeling Reagent, 96 wells

pHSense Eu SNAP labeling reagent assay protocol

The assay begins by culturing cells in a 96-well plate. The pHSense SNAP labeling reagent 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.

pHSense-based detection of GLP1R internalization following Exendin-4 stimulation

Tag-Lite® GLP1R cells (stable cell line, Part #: C1SU1GLP1, Revvity) 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 SNAP Labeling Reagent diluted in cell culture medium (DMEM +10%FBS) were added to the cells, and then incubated for 1 hour at room temperature.

Exendin-4, a GLP1R agonist, was serially diluted in cell culture medium, and 10 µL of each dilution were added to the wells. After incubation at 37°C, the signal was recorded using an HTRF-compatible plate reader. Results show Exendin-4 induced a dose-dependent increase in signal, with an EC₅₀ value consistent with values described in the scientific literature.

The pHSense Eu SNAP Labeling Reagent was tested on 2 different stable cell lines expressing various levels of receptors: Tag-Lite GLP1R cells (HEK-GLP1R) and Tag-lite Mu opioid cells (CHO-MOR).

The comparison of receptor expression levels was performed 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, and then incubated 1h 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 show Tag-Lite GLP1R cells exhibit 5-times higher expression levels than Tag-lite Mu opioid cells.

To perform pHSense Eu SNAP Labeling Reagent 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 incubated overnight at 37°C with 5% CO₂.

After cell supernatant removal, 40 µL of the pHSense Eu SNAP Labeling Reagent diluted in cell culture medium were added to the cells, and then 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 pHSense Eu SNAP Labeling Reagent and receptor agonists prepared in Tag-lite SNAP/CLIP Labeling Medium as physiological buffer.

As demonstrated here, pHSense Eu SNAP Labeling Reagent 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.