Green cADDis cAMP Assay Protocol

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About cADDis cAMP Assays

Cyclic AMP is an essential second messenger for many cellular processes. The messages carried by cAMP are tightly regulated within cells. These assays offer an alternative to measurements of cAMP accumulation in cell lysates, because they detect cell signaling in living cells and can be targeted to specific populations of cells in mixed cultures. The fluorescent sensors used in the assays described here, use one fluorescent protein, so they can be combined with different colored sensors to measure multiple signals simultaneously.

Depending upon the sensor in your kit, the fluorescence can either increase or decrease in response to receptor activation. For example, D0200G cADDis decreases in fluorescence when cAMP levels increase in the cell. One can imagine applications where a downward sensor might have advantages over an upward sensor, for example, if background fluorescence is high. Both Upward and Downward cADDis are bright, robust, and easy to detect on fluorescence plate readers. No need to fix or lyse your cells. Life is interesting!

Overview

The following protocol is optimized for measuring cAMP responses in rapidly dividing, immortalized cell lines on a 96-well plate, and has been validated in live CHO, NIH 3T3, and HEK 293 cells [Graham FL, 1977]. This assay is very robust and can be used for live-cell
imaging or for screening on automated fluorescence plate readers. For use in iPSC-derived or
adherent cells, see Suggestions for Assays in Adherent Cells section
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The BacMam vector carrying these sensors is a modified baculovirus, which can be used for delivery to, and expression in, a wide variety of mammalian cell types including primary cultures.

Materials in the Kit
  • cADDis cAMP sensor BacMam ⩰2 x10*10VG/mL in TNM-FH Insect Culture Medium (Allele Biotech product #ABP-MED-10001)

Green fluorescent sensors that change in fluorescence intensity in response to increases in cAMP. VG/mL is the number of viral genes per milliliter, as distinct from plaque forming units (PFU), that for baculovirus, would be measured in insect cells.

  • Sodium Butyrate (Sigma Aldrich product # B5887) 500 mM in H2O.

Sodium Butyrate is added to the culture to maintain BacMam expression. Other HDAC inhibitors may work as well.

  • β2 Adrenergic Receptor BacMam in TNM-FH Insect Culture Medium (Allele Biotech product #ABP-MED-10001).

A Gs-coupled receptor provided as a positive control for the purpose of assay optimization. Contains a separate red fluorescent protein that is targeted to the nucleus.

DAY 1: Transduce and Plate Cells

This protocol is optimized for rapidly dividing, immortalized cell lines. However, the protocol can be adjusted for transducing non-dividing adherent cells such as neurons, islets, cardiomyocytes, and iPSC-derived lines. We recommend that you take the time to optimize the assay for your particular cell type. See our Suggestions for Adherent Cells following this protocol.

Step 1: Prepare cells (Tube A)

  • Detach cells from flask using standard trypsinization protocol. Resuspend cells in complete culture media and determine cell count.
  • Prepare a dilution of cells at your desired concentration* 100 μL of this cell resuspension will be required for a single well in a 96-well plate, so prepare enough of the dilution to seed the desired number of wells in the plate. Let cells sit in hood and move on to  preparation of the viral transduction reaction.

*480,000 cells/mL works well for HEK293 cells.

Example:
For 96wells (1 plate)

100 μL cell suspension (480,000 cells/mL) per well.
100 μL cells x 110 (96 wells + 10% scale) = 11000 μL cell suspension

When preparing the master mix, scale up by 10-15% to avoid coming up short. To seed a 96-well plate, multiply amounts in Step 1 and Step 2 by 110-120.

Step 2: Prepare Viral Transduction Reaction (Tube B)

  • Prepare a 500 mM stock solution of sodium butyrate in sterile water (in your kit).
  • For each transduction reaction (i.e. one well in a 96-well plate), prepare the transduction solution by mixing 20 μL of the Sensor BacMam stock with 0.6 μL of the 500 mM stock solution of sodium butyrate*, 5 μL of the Receptor control, and 24.4 μL of the complete culture media for your cells, for a total volume of 50 μL. Mix gently.
*Concentration of sodium butyrate should be 6mM in this step. Following Step 3, final concentration of sodium butyrate will be 2mM

Example:
96 wells needed (1 plate). The number of wells desired,  must correspond to the number in Step 1 above.

20 μL Sensor x 110 wells= 2200 μL
5 μL Receptor Control x 110 wells = 550 μL
0.6 μL 500 mM Sodium Butyrate x 110 wells = 66 μL
24.4μL Complete Media x 110 wells = 2684 μL
50 μL total volume per well x 110 wells = 5500 μL transduction mix (96 wells)

Step 3: Mix Cells and Transduction Mix from above.

  • Mix Tube A and Tube B (100 μL tube A + 50 μL tube B). Mix gently and seed 150 μL of mix per well on the 96-well plate.
  • Cover plate with aluminum foil to protect from light and incubate at room temperature for 30 minutes.
  • Incubate ≈24 hrs under normal cell growth conditions, protected from light.

Example:
96 wells needed (1 plate)

100 μL cell suspension per well x 110 wells = 11000 μL master mix
50 μL transduction reaction x 110 = 5500 μL master mix
150 μL total volume per well x110 = 16,500 μL total reaction volume

DAY 2: Measuring Fluorescence

Prior to measuring fluorescence, replace culture media with DPBS. Wash gently so as not to dislodge cells. Cover the cells and allow them to rest at room temperature in DPBS for 25-40 minutes before measuring fluorescence so they can adjust to their new environment
Experiments are performed at 25°Cusing standard GFP excitation and emission wavelengths.

Fluorescence Detection
Our assays are compatible with automated fluorescent plate readers. Partial list of validated instruments:

  • Hamamatsu FDSS
  • Molecular Devices FLIPR
  • Molecular Devices Flexstation
  • Perkin Elmer Enspire
  • Biotek Synergy MX
  • Biotek Cytation
  • BMG CLARIOstar
  • Epifluorescence microscopes

Fluorescence Properties
cADDis is constructed with the very bright, mNeon green fluorescent protein. We recommend Chroma’s Catalog set #49003 for optimal results. Excitation max: 506nm Emission max: 517nm

 

Timing
Unlike many assays that measure accumulation of cAMP in cell lysates, the cADDis assay measures cAMP in living cells, in real time.
For best results, be sure to capture changes in cAMP during the peak response. In Figure 2, fluorescence was captured from cells before the addition of the drug and then sampled at regular intervals. The maximal response is reached at ~360 seconds after the addition of the drug.

The figure below is HEK 293 cells transduced with 20 μL of BacMam carrying green downward cAMP biosensor, activated with isoproterenol. The graph shows the expected timing and duration of cAMP when using the positive controls provided in your kit.

Assay Performance Considerations

How we measure the infectivity of the viral stock:

Typically, viruses are quantified in terms of plaque forming units (PFU). In the case of BacMam, this would be a measurement of the viruses that are capable of transducing an insect cell, the natural host. Since mammalian cell expression is the goal for this assay, we quantify infectivity by measuring viral genes (VG) per milliliter (mL) of the BacMam stock. We use primers specific to the VSVG gene present in the BacMam genome. Viral samples are prepared to release viral genomic DNA, then multiple dilutions of the preparation are run in qPCR against a standard curve to generate an average titer for each viral stock. Check the label on the tube to find VG/mL for your cAMP sensor stock.

Level of sensor expression:

To optimize the assay in your particular cell type, it is important to optimize the amount of virus used in the transduction. Too little virus will produce variable results, particularly if the sensor expression levels are low and difficult to detect on your instrument. In the case of HEK 293 cells, the baseline fluorescence goes up as you add more virus, and when a particular threshold is reached the absolute change in sensor fluorescence, as well as the Z’ for the assay, becomes constant.

Level of receptor expression:

The magnitude of the sensor response can be affected by the level of GPCR expression in your cells. We have found that low levels of receptor expression produce the largest signals, while high levels of receptor expression often produce smaller responses. This is consistent with the observation that over-expression of some GPCRs can change the levels of second messengers due to low levels of spontaneous activity.

Our green fluorescent sensors under CMV promoter control have been validated on several automated fluorescence plate readers. However, sensors under synapsin or weaker promoters, may be limited to detection to imaging systems. In this section, we summarize some of the considerations in the use of various types of fluorescence detection instruments and the trade-offs between these systems.

The simplest format: one drug, one sensor, one time point:

By following the sample protocol described on the previous pages, you can expect to record a robust response on standard fluorescence plate readers. Fluorescence may be recorded sequentially from each well before adding drug, and again after adding the drug. The response to the receptor and agonist controls provided in your kit occurs over a few minutes. While this protocol is simple, the drawback is that it does not capture the kinetics of the response, only the fluorescence before and after the addition of drug.

Two channel format: one drug, two sensors, one time point:

By combining green and red sensors, one can simultaneously record two limbs of second messenger signaling. In this case, standard fluorescence plate readers can be used, but they need the optics necessary to collect two different channels of fluorescence, which will involve specific filter sets and either two detectors or very fast filter switching.

Capturing the kinetics, one well at a time:

High content imaging systems offer excellent opportunities for capturing kinetic responses. In the models that have onboard liquid handling, fluorescence can be recorded before and after the addition of vehicle, and then continuously recorded after addition of the drug. This provides kinetic data, but each well in the plate is recorded over relatively long time frames. Confocal systems are not recommended, as the signal outside of the focal plane is eliminated in these systems.

Capturing the kinetics in parallel, multi-well recording:

There are instruments that can collect fluorescence data from all of the wells of a plate simultaneously and have automated liquid handling. These are perfectly suited for recoding the kinetics of the response from every well. Because the recordings are all done in parallel, the speed of the assay is considerably faster than a single channel instrument. An example of such an instrument is the Molecular Devices “Flex Station” and “FLIPR” series of plate readers and our assays have been validated on these instruments.

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Storage/Biosafety/Warranty

BacMam stocks should be stored at 4°C and protected from light. Avoid repeated freeze/thaw cycles.

Additional Materials not Supplied

-Greiner CellCoat (#655946) is our preferred 96-well plate available from VWR.
-Dulbecco’s Phosphate Buffered Saline (DPBS) available from VWR [Dulbecco, R. and Vogt, M.1957].

BioSafety Considerations

BacMam is a modified baculovirus, Autographa californica, AcMNPV. The virus in this kit is pseudotyped to infect mammalian cells.
In mammalian cells, the baculovirus genome is silent, and it cannot replicate to produce new virus in mammalian cells. While it should be handled carefully, in a sterile environment, it is classified as a Biosafety Level 1 (BSL-1) reagent.
This product is for research use only and is not recommended for use or sale in human or animal diagnostic or therapeutic products.

Warranty

Materials are provided without warranty, express or implied. End user is responsible for making sure product use complies
with applicable regulations. No right to resell products or any components of these products is conveyed.

Trouble Shooting

Need some help? Call us at 406-595-4725 or email info@montanamolecular.com  Also, here are a few simple trouble shooting steps:

Are your cells fluorescent?
Different types of promoters drive expression in mammalian cells. The CMV promoter in our BacMam vectors is an effective promoter in many cell lines, but not all. Twenty four hours after transduction, you should see bright green fluorescent cells in a typical epifluorescence microscope, or the transduced wells in a 96 well plate should be significantly more fluorescent than untransduced cells in wells on the same plate.

HDAC inhibitors are important to expression of the sensors. While BacMam transduction alone will initially generate low levels of sensor expression, sodium butyrate or another HDAC inhibitor such as VPA or trichostatin A (TSA) will generate optimal levels of sensor expression and maintain this level of expression [Kost, T. et. al. 2007] If cells look unhealthy, lower concentrations of HDAC inhibitor may be used. This may improve cell health, but it will also reduce sensor expression.

Finally, the type of cell culture media used in your experiment can affect the transduction efficiency of BacMam. Our assays have been validated in EMEM, DMEM, and F12K culture media.

Is the positive control working?
If the cells are expressing the sensor, and fluorescence is detectable on your instrument, then check the sensor using the positive control included in this kit. Adding 5 μL of the β2 adrenergic receptor virus to a set of control wells will ensure that a Gs-coupled receptor is present in all of the cells. You can double check to make sure the receptor is expressed by examining the cells in a fluorescent microscope with filters for red fluorescence. You should see the green sensor fluorescence throughout the cell, and red nuclear fluorescence that marks the cells that also express the β2 adrenergic receptor.

Addition of isoproterenol will cause a change in fluorescence. If it does not, then it is important to use this positive control to optimize three aspects of your assay (see Figure 3). First, a serial dilution series of the sensor with a constant amount of receptor virus can be used to find the optimal sensor expression for your instrument and cell line. Second, it is important that you find the amount of virus sufficient to transduce all of the cells in the well. Third, it is important to determine what the kinetics of the response is and whether your instrument can measure in the appropriate time frame.

Figure 3.
As the amount of cADDis added to the
wells increases, so does the baseline fluorescence,
plotted in purple. The change in fluorescence when
cAMP changes also increases with more virus, but
reaches the maximum possible change and remains
constant over a broad range of virus concentration
(circles). Z’ is high over a broad range of virus
concentration (triangles).

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