Real-Time Monitoring of Odorant Receptor Activation in Recombinant Cells

Abstract

Source: de March, C. A. et. al., Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase. J. Vis. Exp. (2019)

This video demonstrates the method to monitor the real-time activation of odorant receptors in recombinant mammalian cells using luminescence measurement. Odorant binding triggers G-protein activation and cAMP production, activating glosensor proteins that emit luminescence upon substrate binding, providing a quantitative measure of receptor response.

Protocol

1. Hana3A Cells Culture

1. Prepare M10 (Minimum Essential Medium (MEM) plus 10 % v/v fetal bovine serum (FBS)) and M10PSF (M10 plus 100 µg/mL penicillin-streptomycin and 1.25 µg/mL amphotericin B).
2. Culture the cells in 10 mL of M10PSF in a 100 mm cell culture dish in an incubator set at 37 °C and 5% carbon dioxide (CO2).
3. Divide the cells every 2 days at a 20% ratio: when 100% confluence of cells (approximately 1.1 x 10⁷ cells) is observed under a phase-contrast microscope, aspire the media and wash the cells gently with 10 mL of phosphate-buffered saline (PBS).
4. Aspirate PBS and add 3 mL of 0.05% trypsin-ethylene diamine tetraacetic acid (EDTA, 0.48 mM). Let act for approximately 1 minute, until the cells dissociate from the plate.
5. Add 5 mL of M10 to inactivate the trypsin and eventually detach the cells still attached to the plate by pipetting up and down.
6. Transfer the volume (8 mL) to a 15 mL tube and centrifuge at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells into 5 mL of M10PSF by pipetting up and down to break any cell mass. Avoid creating bubbles in the tube.
7. Transfer 1 mL of the resuspended cells solution in a new 100 mm cell culture dish and add 9 mL of fresh M10PSF. Incubate at 37 °C and 5% CO₂.

2. Preparation of the Cells for Transfection

1. Evaluate the confluence, or the number of cells, by observing them under a phase-contrast microscope. At least 10% confluence (approximately 1.1 x 10⁶ cells) is needed for one plate.
2. Aspirate the media and wash the cells gently with 10 mL of PBS. Aspirate PBS and add 3 mL of EDTA. Let act for approximately 1 min at room temperature, until the cells dissociate from the plate.
3. Add 5 mL of M10 to inactivate the trypsin and eventually detach the cells still attached to the plate by pipetting up and down. Transfer the volume (8 mL) to a 15 mL tube and centrifuge at 200 x g for 5 min.
4. Aspirate the supernatant and resuspend the cells into 5 mL of M10PSF by pipetting up and down to break any cell mass. Avoid creating bubbles in the tube.
5. Depending on the number of plates to be transfected, transfer an appropriate amount of cells in a reservoir with the proper corresponding volume of M10PSF. One 96-well plate should be plated with 1/10 of a 100% confluent 100 mm dish (approximately 1.1 x 10⁶ cells) diluted in M10PSF to reach a total volume of 6 mL. For one 96-well plate starting with a 100% confluence 100 mm dish, add 500 µL from the 5 mL of resuspended cells to 5.5 mL of fresh M10PSF. Mix the cells and M10PSF without generating air bubbles.
6. Pipette 50 µL of the suspended cells into each well of the 96-well plate using a multichannel pipette. Incubate overnight at 37 °C and 5 % CO₂.

3. Plasmid Transfection

1. Observe the 96-well plate under a phase-contrast microscope to ensure a cell confluence between 30% and 50%.
2. Prepare a first transfection mix that contains the plasmids common to the entire plate (a shorter version of receptor-transporting protein 1 (RTP1S), odorant receptor (OR), and Glosensor protein; see Table of Materials) following the volumes in Table 1. Notice that the quantity of Rho-tagged OR should be divided by the number of ORs if several ORs.
   NOTE: We strongly advice to add an empty vector negative control (here Rho-pCI) and any positive control (OR known to respond to the tested odorant) to the experiment plan.
3. Prepare a second transfection mix containing 500 µL of MEM and 20 µL of Lipofectamine 2000 reagent (valid for one 96-well plate, see Table of Materials). Add the second mix to the first one, and gently mix by pipetting up and down and incubate for 15 min at room temperature. Add 5 mL of M10 and mix gently.
4. Replace the M10PSF in the previously platted 96-well plate by 50 µL of the final transfection media. Incubate in an incubator set to 37 °C and 5% CO₂ and vacuum the chamber of the luminometer overnight.

4. Substrate Incubation

1. Observe the 96-well plate under a phase-contrast microscope to ensure a cell confluence between 60% and 100%. Prepare a simulation solution of Hank’s Balanced Salt Solution (HBSS) containing 10 mM of hydroxyethyl piperazineethanesulfonic acid (HEPES) and 5 mM of D-glucose.
2. Dilute 75 µL of the cyclic adenosine monophosphate (cAMP) reagent (see Table of Materials) solution to 2.75 mL of the stimulation solution. Remove the transfection medium from the 96-well plate and wash the cells by adding 50 µL of fresh stimulation solution to each well.
3. Remove the stimulation solution and add 25 µL of cAMP reagent solution prepared in step 4.2 to each well. Incubate the 96-well plate at room temperature in a dark and odor-free environment (for example, a clean empty drawer far away from chemicals or any odorant source) for 2 h.

5. Odorant Stimulation

1. First, equilibrate the luminometer chamber with volatile odorant molecules. Dilute the odorant to the desired concentration in 10 mL of mineral oil (Figure 1A). Before the end of the cAMP reagent incubation time, add 25 µL of the odorant solution to a new 96-well plate (not the one containing the cells). Incubate this odorant plate at room temperature in the luminometer chamber for 5 min (Figure 1B) (no luminometer recording is required here).
2. Set the luminometer to record the luminescence with 0 s of delay during 20 cycles of plate measurement of 90 s with 0.7 s of interval between cycles.
3. Right before reading the plate, remove the odorant plate from the chamber. Add 25 µL of odorant between the wells of the 96-well plate containing the cells (do not add the odorant in the wells containing the cells) and quickly start the luminescence measurement of all wells for 20 cycles within 30 min (Figure 1C).

Table 1: Mix for transfection. Quantities of plasmids (Glosensor protein, RTP1S and OR) to add to MEM to transfect to one 96-well plate.

	Per 96-well plate
MEM	500 µL
pGlosensor	10 ng
RTP1S	5 ng
OR	75 ng

Results

Figure 1: Schematic protocols for real-...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05 % trypsin-EDTA	Gibco	25300-054	0.05% Trypsin - EDTA (1x), phenol red - store at 4 °C
100 mm cell culture dish	BD Falcon	353003	100 mm x 20 mm cell culture dish
15 mL tube	BD Falcon	352099	17 mm x 120 mm conical tubes
96-well plate	Corning	3843	96 well, with LE lid white with clear bottom Poly-D-lysine coated Polystyrene
Amphotericin	Gibco	15290-018	Amphotericin B 250 µg/mL - store at 4 °C
Centrifuge machine	Jouan	C312	Centrifuge machine with swinging bucket rotor for 15 mL
Class II Type A/B3 fumehood	NUAIRE	NU-407-500	Fumehood for cell culturing
FBS	Gibco	16000-044	Fetal Bovine Serum - store at -20 °C
GloSensor cAMP Reagent	Promega	E1290	GloSensor cAMP Reagent luminescent protein substrate - store at -20 °C
Incubator 37 °C; 5 % carbon dioxide	Fisher Scientific	11-676-604	Incubator for cell culturing
Lipofectamine 2000 reagent	Invitrogen	11668-019	Lipofectamine 2000 Reagent 1mg/ml transfection reagent - store at 4 °C
Luminometer POLARstar OPTIMA	BMG LABTECH	discontinued	96 well plate reader for luminescence
Mineral oil	Sigma	M8410	Solvent for odorants - store at room temperature
Minimum Essential Medium (MEM)	Corning cellgro	10-010-CV	Minimum Essential Medium Eagle with Earle’s salts & L-glutamine - store at 4 °C
Penicillin/Streptomycin	Sigma Aldrich	P4333	Penicillin-Streptomycin solution stabilized with 10,000 U of penicillin and 10 mg streptomycin - store at -20 °C
pGlosensor	Promega	E2301	pGloSensor-22F cAMP luminescent protein plasmid - store at 4 °C
Phase contrast microscope	Leica	090-131.001	Phase contrast microscope with x4, x10, x20 objectives
RTP1S	H. Matsunami lab	-	100 ng/µL plasmid - store at 4°C