TITLE: Monitoring cell-surface expression of GPCR by ELISA
AUTHORS: Elie Besserer-Offroy, Rebecca L Brouillette, Jean-Michel Longpré, Philippe Sarret

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Quantifying cell surface expression of G Protein-Coupled Receptors (GPCRs) can be exteremly important for the expression of mutant receptors. Herein we report a useful Enzyme-Linked Immunosorbent Assay (ELISA) for the cell-surface detection of a HA-tagged version of a GPCR.</div></div>

[STEPS]
?. [Day 1 - Cell Culture & Transfections]
Coat 24-well plates with Poly-L-Lysine (this need to be done in a biological safety cabinet to ensure sterility).
?. [Day 1 - Cell Culture & Transfections]
Add  of  Poly-L-Lysine solution in each well of the 24-well plate and incubate  at .This can be done using a 300µL multichannel pipet fitted with 4 tips
300 µl
0 Room temperature
?. [Day 1 - Cell Culture & Transfections]
Remove the Poly-L-Lysine solution (this solution can be re-used up to 4 times to coat cell culture plasticware).This can be done using a 300µL multichannel pipet fitted with 4 tips
?. [Day 1 - Cell Culture & Transfections]
Rinse the wells twice with  of sterile water.
300 µl
?. [Day 1 - Cell Culture & Transfections]
Let dry the 24-well plate without lid under the biological safety cabinet for  before seeding cells.
Poly-L-Lysine-coated plates can be stored for several weeks at   befor use.
?. [Day 1 - Cell Culture & Transfections]
Prepare transfections of plasmids encoding HA tagged-GPCRs (this need to be done in a biological safety cabinet to ensure sterility).For 3 well of the 24-well plate:
In the case you want to compare expression of a mutant receptor, positive and negative controls are needed to normalize the results. The positive control should be the wild-type receptor and the negative control should be cells transfected with the empy vector (MOCK cells).
?. [Day 1 - Cell Culture & Transfections]
Add  of Opti-MEM into  a sterile 5mL tube.
300 µl
?. [Day 1 - Cell Culture & Transfections]
Prepare HEK293 cells for transfection (this need to be done in a biological safety cabinet to ensure sterility).
Ideally, cells were seeded at a density of 3 million cells per 10cm-pretri dish 48h before transfection to ensure a high transfection rate.
?. [Day 1 - Cell Culture & Transfections]
Add  of plasmidic DNA encoding for the desired HA tagged-GPCR to the tube containing Opti-MEM and mix.
1.5 µg
?. [Day 1 - Cell Culture & Transfections]
Add  of P3000 Reagent to the tube and mix.
3 µl
?. [Day 1 - Cell Culture & Transfections]
Add  of Lipofectamine 3000 to the tube, mix, and incubate at  for .
2.25 µl
0 Room temperature
?. [Day 1 - Cell Culture & Transfections]
Remove culture media and rinse cells with PBS.
?. [Day 1 - Cell Culture & Transfections]
Add  of 0.25% Trypsin to a 10-cm petri dish and incubate for   at .
1 ml
37 °C
?. [Day 1 - Cell Culture & Transfections]
Add  of complete DMEM (10% FBS, 20mM HEPES, Penicilin/Streptomycin) to the petri dish and dissociate cells by pipeting up and down.
5 ml
?. [Day 1 - Cell Culture & Transfections]
Count cells using an automated cell counter or a hemacytometer.
?. [Day 1 - Cell Culture & Transfections]
Adjust cell concentration to 150,000 cells/mL.
?. [Day 1 - Cell Culture & Transfections]
Add  of the cell suspension at 150,000 cells/mL to the 5mL tube containing the plasmidic DNA and mix gently.
2 ml
?. [Day 1 - Cell Culture & Transfections]
Dispense  of the mix of cell and plasmidic DNA to the desired wells of the Poly-L-Lysine-coated 24-wellplate.
450 µl
?. [Day 1 - Cell Culture & Transfections]
Incubate at  for .
[in humidified chamber at 5% CO2]
?. [Day 3 - ELISA]
Detection of cell surface expression by ELISA (this part of the protocol can be done on the wet bench).
Buffer and reagents in this part of the protocol can be dispensed to the 24-well plate using combitips and a single channel repeater.
?. [Day 3 - ELISA]
Remove cell culture media and wash each well with  of PBS.
400 µl
?. [Day 3 - ELISA]
Fix cells using  of  formaldehyde in TBS for  at .
400 µl
0 Room temperature
?. [Day 3 - ELISA]
Rinse cells three times with  of TBS.
500 µl
?. [Day 3 - ELISA]
Block non-specific sites using  of  non-fat dry milk disolved in TBS for under gentle orbital agitation.
400 µl
In case non-fat dry milk is not suitable for blocking non-specific sites, a solution of   of Bovine Serum Albumine in TBS can be used. The same solution must also be used for the incubation of the antibody.
?. [Day 3 - ELISA]
Remove blocking solution and add  of 1/1000 dilution of HRP-linked anti-HA antiboby diluted in  non-fat dry milk in TBS for  under gentle orbital agitation.
250 µl
This incubation step can also be done   at   under gentle orbital agitation.
?. [Day 3 - ELISA]
Remove antibody solution and wash each well three times with  of TBS.
500 µl
?. [Day 3 - ELISA]
Add  of  3, 3′,5 ,5′-Tetramethylbenzidine (TMB) Liquid Substrate, Supersensitive, for ELISA and incubate under gentle orbital agitation for 2 to 15 min (until the color of your positive control turn intense blue).
250 µl
0 Room temperature
?. [Day 3 - ELISA]
Stop the TMB reaction by adding  of  Hydroclhoric Acid (HCl).
250 µl
?. [Day 3 - ELISA]
Transfer  of the colorimetric reaction to a flat-bottom transparent 96-well plate.
100 µl
?. [Day 3 - ELISA]
Read the absorbance at 450nm using a multimode plate reader.
?. [Results analysis]
As this quantification of cell surface expression is a semi-quantitative method it should not be presented as raw OD450nm values but rather as a percentage of expression compared to the positive control (or wild-type receptor).To normalize the results, average the OD450nm of the positive control and the OD450nm of the negative control and apply the following formula: