Red DAG Assay Protocol

About These Assays #D0300R Red Down DAG and #U0300R Red Up DAG

Many cell surface receptors couple to the heterotrimeric G protein Gq, which in turn activates phospholipase C (PLC). PLC produces two different second messengers, diacylglycerol (DAG) and inositol triphosphate (IP3), causes an increase in Ca2+. This coordinated increase of both DAG and Ca2+ triggers the activation of conventional protein kinase C (cPKCs) to phosphorylate many different protein targets. Live cell assays that measure increases in Ca2+  have been used for many years to detect this pathway, but a rise in Ca2+ is an ambiguous signal: there are other signaling pathways that cause increases in intracellular Ca2+. This assay for DAG can be used to unambiguously resolve PLC pathway activation in living cells, and it can be combined with a green fluorescent sensor for  Ca2+  or PIP2 to better understand the kinetics of these coordinated, parallel signaling processes [Tewson P, et.al. 2012].

Depending on the kit, fluorescence either increases or decreases in response to activation.  The Downward DAG sensor (#D0300R) decreases in fluorescence.  One can imagine applications where this sensor might have advantages over a sensor that increases in fluorescence, for example, if background fluorescence is significant, the decreasing fluorescence from the sensor can be more easily separated from background fluorescence.

Overview

The red fluorescent DAG sensors, are used for measuring diacylglycerol changes in live mammalian cells. This protocol is optimized for imaging live cells on a 96-well plate and has been validated in HEK293, CHO and NIH 3T3 cells. These sensors are suitable for live-cell imaging and for screening on automated fluorescence plate readers. This protocol can be adjusted and optimized for many different types of cells, including primary cultures, iPSC-derived cells, and pancreatic islet cells, and is easily scaled for 384 well format. Because these are single fluorescent protein sensors they can be combined with a green fluorescent protein or green fluorescent DAG sensor for ratiometric imaging.

The vector carrying these sensors is a modified baculovirus. In mammalian cells, BacMam expresses only the fluorescent sensor and is a BSL-1 reagent.  If you want to measure DAG in cells other than HEK 293, then the VG/mL needed to transduce a well of HEK 293 cells can be used as a starting point for assay optimization in other cell types. We recommend that you take the time to do a dilution series of the sensor in your cells, to optimize in your particular cells and for your fluorescence detection system.  If your cells work best for a sensor other than CMV, please let us know.

Materials in the Kit

DAG sensor in BacMam in TNM-FH Insect Culture Medium  (Allele Biotech product #ABP-MED-10001).
-Red fluorescent sensor that changes in response to increases in DAG. Downward sensors decrease in fluorescence following activation of a Gq-coupled GPCR. Upward sensors increase following activation of a Gq-coupled GPCR. VG/mL are units that produce expression in mammalian cells, as distinct from plaque forming units (PFU).

Sodium Butyrate (Sigma Aldrich product number B5887) 500 mM.
-Sodium Butyrate is added to the culture to maintain BacMam expression.  Other HDAC inhibitors may work better in other cell types.  See FAQs on montanamolecular.com

hM1 muscarinic acetylcholine receptor BacMam in TNM-FH Insect Culture Medium (Allele Biotech product #ABP-MED-10001).
-A Gq-coupled GPCR in a BacMam vector, is a positive control. This vector separately expresses a green fluorescent protein that is targeted to the nucleus.

Carbachol 25 mM in H2O
-Carbachol is used to stimulate Gq signaling through the positive control receptor.

Additional Materials Not Supplied
-Greiner CellCoat (#655946) is our preferred plate for this assay.
-Dulbecco’s Phosphate Buffered Saline [2].
-Complete culture media specific to your cells. (Please see Assay Considerations Section) 

Storage/Biosafety/Warranty

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

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 or reverse-engineer products or any components of these products is conveyed.

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*. 100μL 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.

*420,000-440,000 cells/mL works well for HEK293 cells.

Example:
For 96 wells (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 10-15 μL of the Sensor BacMam stock with 0.6 μL of the 500 mM stock solution of sodium butyrate*, 5 μL of Receptor control, and the appropriate amount of 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.

15 μL Sensor x 110 wells = 1650 μL
5 uL Receptor Control x 110 wells = 550uL
0.6 μL 500 mM Sodium Butyrate x 110 wells = 66 μL
29.4 μL Complete Media x 110 wells = 3234 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 then 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. Return plate to incubator.
  • Incubate for ≈ 36-48 hrs under normal cell growth conditions, protected from light. 24 hr incubation periods may be used, but red fluorescence will be much lower.

** 1 mM sodium butyrate may improve cell health but also reduce fluorescence.

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 Incubation

  • Red fluorescent sensors can take several hours longer to mature than their green fluorescent cousins.
DAY 3: Measuring Fluorescence

Day 2 Incubation 

Red fluorescent sensors can take several hours longer to mature than their green fluorescent cousins.

Day 3 MEASURING FLUORESCENCE

  • Cells are now ready for assay. 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 20-30 minutes before measuring fluorescence.  Experiments are performed at 25°C . The optimal excitation wavelength is 590 nm. A broad band pass emission filter spanning 600-700 nm is ideal. 
  • 30 μM carbachol (included in the kit) can be used to activate control wells transduced with M1 receptor control.
  • This red fluorescent sensor has been validated on a variety of epifluorescence microscopes with lenses ranging from 20 X, 0.9 N.A. to 63X, 1.4 N.A.  Before deploying in automated plate readers, we recommend that you optimize the assay on a microscope. The following sections provide more detail on automated measurement.

Fluorescence Properties

The optimal excitation wavelength is 590 nm, but the absorption band of this protein is quite broad, so broad bandpass filters that pass 550 to 590 nm light can be used quite effectively.  On the emission side, the red light spans 600 to 700 nm, so broad band pass emission filters can also help to collect much of the emission.

 

Timing

This is a live cell assay, and unlike many assays that measure accumulation of an analyte over time, this sensor detects, in real time, the DAG level in the living cell.  Changes in DAG can occur quite rapidly, so the application of drug and resulting changes should be captured as quickly as possible.  The best possible experimental setup involves capturing the fluorescence from a well of cells first, before the addition of any compound, and then again a second or so after the compound addition.  Sampling the fluorescence at intervals of 2 to 5 seconds provides a good measurement of the response. In HEK293 cells, the maximal response using the control receptor is reached at ~ 60 seconds after the addition of the agonist.

Assay Performance Considerations

Virus Titer

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

Level of sensor expression

To optimize the assay for your particular cell type, it is important to titrate 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. Too much virus, on the other hand, can reduce the magnitude of the sensor signal (i.e. the change in fluorescence that results from sensor activation).

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 often 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.

Ratiometric Measurement

The red fluorescence emitted by the red cADDis sensor is not as bright as the green cADDis sensors.  Because the red fluorescent sensor is not as bright, it may not be suitable for use alone on some imaging systems. However, red cADDis can be combined with green cADDis for ratiometric imaging applications as shown in Figure 2.

Culture Media Considerations 

This assay has been validated with EMEM, DMEM, and F-12K complete growth media. Other types of media may affect results. 

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

We have validated this assay on BioTek Synergy MX and BMG CLARIOStar fluorescence plate readers using the protocol described on the previous pages. Control receptor agonists are added by hand, and then the plate is inserted into the fluorescence plate reader to record the fluorescence from each well sequentially. This can all be done by hand because these sensors respond over several minutes to agonists in the well. While this protocol is simple, the drawback is that it does not capture the kinetics of the response, simply the sensor fluorescence before and after the addition of drug. Automated drug dispensers can be used, but care must be taken to make sure that the dispenser does not blast the cells 

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 to be fitted with 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. Most two channel plate readers can scan quickly, but the kinetics of the responses are lost.  This can be quite limiting if the kinetics of the two second messenger systems are different.  For example, the Gq stimulated release of Ca2+ stores can be quite rapid, while the DAG signaling occurs over a longer time frame.  If the timing is off with a single point measurement, the DAG signaling might be detected, but without catching the Ca2+ transient.

Capturing the kinetics, one well at a time.

High content imaging systems offer an opportunity 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.  Our assays have been validated on the BioTek Cytation imaging system. However, the red fluorescent assays may not be suitable for use on some automated confocal-based systems because only the signal on the focal plane is collected on confocal instruments.

Troubleshooting

Are the 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. Do you know that the CMV promoter works in your cell line? Twenty-four to forty-eight hours after transduction, you should see red 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 valproic acid (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.

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. Finally, if there is a specific promoter system that you know drives efficient expression in your cells, please let us know.  We are continually adding new promoter systems to our sensors and we may already have the one you want to use. 

Is the positive control working?

If the cells are expressing the sensor, and fluorescence is detectable on your instrument, then a good positive control is included in this kit.  Adding 5 μL of the M1 muscarinic acetylcholine receptor virus to a set of positive control wells will ensure that a Gq-coupled receptor is present.  You can double check to make sure the receptor is expressed by examining cells in a fluorescent microscope with filters for green fluorescence.  You should see the red sensor fluorescence throughout the cell, and green nuclear fluorescence that marks the cells that also express the receptor control.

Addition of carbachol will then generate a change in fluorescence. If it does not, then it is important to use this positive control to optimize three aspects of your assay. First, a serial dilution series of the sensor with a constant amount of receptor virus can be used to optimize for your cells, receptor, and instrumentation. Second, 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 assay and instrument are capable of making the measurements in that time frame.

Contact Us!

After following the suggested protocol and trouble shooting steps, if you still have questions or feedback please let us know. We strive to respond to emails sent to [email protected] within 24 hours.

References

References

  1. U.S. Patent No. 9,547,017. Genetically-encoded fluorescent sensors for detecting intracellular signaling through diacylglycerol pathways.
  2. Graham FL, Smiley J, Russel WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen Virol. 1977, 36(1):59-74.
  3. Dulbecco R Vogy, M. Plaque formation and isolation of pure lines with poliomyelitis viruses. The Journal of Experimental Medicine. 1954.
  4. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science 1994.
  5. Kost T, Condreay J, Ames R, Rees S, Romanos M. Implementation of BacMam virus gene delivery technology in a drug discovery setting. Drug Discovery Today 2007, 12(9-10):396-403.
  6. Pending PCT Patent Application EP2825887A1. Genetically-encoded fluorescent sensors for detecting intracellular signaling through diacylglycerol pathways.