Flow Cytometry Assay to Measure Internalization of GPCRs

Internalization of receptor is measured as the reduction of receptors on the plasma membrane surface. Surface receptors are labeled with fluoresent antibodies which is in turn measured using a flow cytometer. The antibodies recognize an epitope engineered onto the N-terminus of the GPCR.

The following protocol is optimized for internalization of FLAG-tagged beta 2-adrenergic receptor (B2AR) stably transfected into HEK293 cells.

Protocol

1) Grow up receptors in 37degC incubator until confluent onto 6-cm dishes.

2) Fill dishes with 2 mL growth media with 30 mM HEPES, pH 7.4. Set aside a dish of 293/B2AR at 4degC. You will use this dish to estimate the total number of receptors on the surface of a sample (i.e. before agonist treatment). HEPES reduces exposure of the cells to extreme pH values outside of the incubator.

3) Add agonist to dishes and place in incubator.

4) At the appropriate time points, remove dishes from incubator and chill cells on ice. NOTE: In order to prevent additional endocytosis of receptors, you must perform all of the following procedures on a bed of ice or in the cold room.

5) Rinse all dishes with ice-cold PBS, including the 293/B2AR sample left on ice.

6) Harvest cells - rinse briefly with ice-cold PBS Ca-free, Mg-free, 0.04% EDTA - add 2.5 mL ice-cold CDB to lift cells.

CDB-Cell Dissociation Buffer, Enzyme-free, PBS-based (Gibco BRL Life Technologies) Our campus flow cytometry facility manager recommended this buffer for lifting cells and reducing cell aggregates. However, the user can probably also substitute PBS Ca-free, Mg-free plus 0.04% EDTA to lift cells.

7) Pellet cells in centrifuge set at 4degC and rinse 1x with media. NOTE: Since the anti-FLAG M1 antibody (Sigma, Covance)requires Ca ions to bind the FLAG epitope, you must rinse the pellet with media to get rid of any residual chelating agents.

8) Label receptors with fluorescent antibody -resuspend pellet in 500 µL of media containing 10 mg/mL FITC-M1 (M1 which has been covalently bound to the FITC fluorophore (Molecular Probes) using a standard procedure) - incubate 45' on ice with occasional agitation to ensure even distribution of antibody.

9) right before analyzing samples on the flow cytometer, add 1 µL of propidium iodide (PI) (Molecular Probes) to each sample to stain dead cells.

10) Use flow cytometry to measure the fluorescence of each sample. The user should consult their local flow cytometry facility for general instruction on flow cytometry and advice on specific procedures for collection and analysis of samples. We typically use a Becton Dickinson FACScan, gate out PI-stained cells, and use the mean fluorescence as a measure of fluorescent antibody associated with the cells. References: Chu et al. (1997)

Chu et al. (1997) J Biol Chem. 272(43):27124-30

Patricia Tsao and Mark von Zastrow, "Type-specific Sorting of G Protein-coupled Receptors after Endocytosis." J Biol Chem. April 14, 2000; 275(15):11130-11140.