Measurement of cAMP fluorescence in HEK293 cells

Real time cAMP assay

Procedure

A. Day 1: Plate cells

1. 18–24 hours before transfection, plate HEK293T cells (2x10⁴/100µl/well) in a 96-well plate (Thermo Scientific™ Nunc™ MicroWell™ 96-Well Optical-Bottom Plates with Polymer Base #12- 566-70) in complete growth medium.
2. Culture cells in a 5%CO₂ + 21%O₂ incubator at 37⁰C overnight.

B. Day 2: Transfection of Olfactory Receptors plasmid in HEK293T cell with TransIT-2020 reagent (Mirus#MIR5400)

The following protocol is for 1 well of a 96-wellplate

1. Warm TransIT-2020 Reagent to room temperature and vortex gently before using.
2. Place 9 µl of DMEM serum free antibiotic free medium in a sterile tube.
3. Add 0.1µg plasmid DNA.
4. Pipet gently to mix completely.
5. Add 0.3µl TransIT-2020 Reagent to the diluted DNA mixture.
6. Pipet gently to mix completely.
7. Incubate at room temperature for 15–30 minutes to allow complexes to form.
8. Add 92 µl culture medium without antibiotic to the TransIT-2020 Reagent: DNA complexes.
9. Aspirate the medium from cells and add the complexes to cells.
10. Incubate cells in a 5%CO₂+21% O₂ incubator at 37⁰C for 4 hours.

C. Transduction of cADDis cAMP sensor BacMam baculovirus (Montana Molecular#U0205G) to HEK293T cells transfected with Olfactory Receptor plasmid.

4 Hours later prepare the Viral Transduction Reaction for 1 well of96-well plate as outlined below:

1. Experimental well- 20 μL Sensor+ 0.6 μL 500 mM Sodium Butyrate+ 25.4μL Complete Media for experimental well:                   Positive sensor control- 20 μL Sensor+ 5 μL Receptor Control+0.6 μL 500 mM Sodium Butyrate+ 25.4μL Complete Media for positive sensor control well.
2. Add 50 μL of transduction reaction to each well containing HEK293T cells transfected with Olfactory Receptor plasmid.
3. Leave one well of the same Olfactory Receptor transfected HEK293T cells with no sensor as control.
4. Incubate cells in a 5%CO₂ +21% O₂ incubator at 37⁰C for 24 hours.

D. Day 3: Measuring fluorescence

1. Prior to measuring fluorescence, replace culture media with 150μL 37⁰C DPBS (Gibco#14040- 133). Wash gently so as not to dislodge cells. Cover cells and allow them to rest at room temperature in DPBS for 25-40 minutes before measuring fluorescence by allowing cells to adjust to new environment.
2. Aspirate the 150 μL DPBS from the cells and add 100 μL DPBS (at room temperature) to each well. Cover the plate with clear Seal Plate film (SIGMA#Z369659-100EA).
3. Concentration-dependent effects of H2S are measured by cAMP fluorescence changes in a microplate reader by setting wavelength of excitation at 495 nm and emission at 540 nm using the instrument (SynergyH1, Biotek, Winooski, VT). After collecting two baseline readings of cAMP fluorescence, add 10 μl of NaHS with various concentrations or 15 mM sodium acetate (NaAce, included in the kit), or 5 μM isoproterenol (ISOP, included in the kit) from stock solutions (see below “preparation of stock solutions”). Record intensity of cAMP fluorescence every minute for 8 minutes. Quantify changes in cAMP fluorescence to a given stimulus using the following equation: ΔF = (F-F₀)/F₀.  Where F is the reading of cAMP fluorescence intensity after the stimulus, F₀ is the reading from the same well before the stimulus was applied, and ΔF representing changes in cAMP fluorescence intensity.
4. Copy the data to excel and analyze the data.

Preparation of stock solutions for cAMP assay:

• Prepare 1.65M Sodium Acetate (SIGMA #S2889) in DPBS and dilute 10 times to 165mM with DPBS. Keep on ice.
• Prepare 55µM Isoproterenol by diluting 10mM Isoproterenol (provided in the kit) with DPBS. Keep on ice (positive control).
• Prepare stock 1.1M NaHS (SIGMA #161527) in degassed DPBS (prepare NaHS solution fresh just before use for each experiment).
• Keep NaHS stock solution on ice. Keep 1ml of DPBS on ice as well for negative control.