Two-Color Cell Stress Assay Protocol

The following protocol is optimized for measuring cell stress responses on a 96-well plate of living cells. It has been validated in live HEK 293 cells as well as in iPSC-derived cardiomyocytes and neurons.  Assay fluorescence can be detected on live-cell imaging systems, automated fluorescence plate readers or fluorescence microscopes. For use in iPSC-derived or adherent cells, see Suggestions for Assays in Adherent Cells section.

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. 

Two-Color Cell Stress sensor BacMam ⩰2 x10¹⁰ VG/mL in ESF 921 Insect Culture Medium (Expression Systems, product # 96-001-01).

• Green fluorescent sensor that increases in fluorescence intensity in response to ER and cellular stress. Constitutively expresses a red fluorescent protein for two-color measurements. 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.

Thapsigargin (Cayman Chemical product # 10522) dissolved in DMSO, diluted to 100 μM in H₂O

• Thapsigargin is a SERCA pump inhibitor that induces high levels of ER stress. It is used as the positive control when conducting the cell stress assay. Thapsigargin is supplied in 20 µL aliquots in airtight tubes purged with nitrogen. Thapsigargin will arrive on ice. It should immediately be aliquoted and stored at -20°C. On the day of the experiment an aliquot may be thawed and diluted into H₂O or DPBS buffer and used immediately. Discard any unused thapsigargin aliquot.

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].
• Optional: FluoroBrite DMEM media (ThermoFisher Scientific product # A1896701) supplemented with 10% FBS and 4 mM GlutaMAX.

This protocol is optimized for rapidly dividing, immortalized cell lines in 96 well plates. 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 Other Cell Types and Plate Formats following this protocol.

 

Step 1: Prepare cells (Tube A)

• Detach cells from flask using standard trypsinization protocol. Re-suspend 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.

* 500,000 cells/mL works well for HEK293T cells. This will result in 50,000 cells/well in a 96-well plate. Optimal cell density is cell type dependent.

Example: 96 wells needed (1 plate).

100 μL cell suspension (500,000 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 25 μL of the Sensor BacMam stock with 0.6 μL of the 500 mM stock solution of sodium butyrate*, 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.

25 μL Sensor x 110 wells= 2750 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 ≈ 20-24 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 wells = 5500 μL master mix
150 μL total volume per well x110 = 16,500 μL total reaction volume

To set up the assay in 384-well plates, follow all of the protocol steps outlined above, adjusting reagent volumes as follows:

Step 1) Prepare cells (Tube A)

• 50 μL of the cell resuspension will be required for a single well in a 384-well plate. A plating density of 12,500 cells per well is a good starting point, so prepare the cell suspension at 250,000 cells/mL. Depending on the cell type and plate type, 10,000-15,000 cells per well may be optimal.

Step 2) Prepare Viral Transduction Reaction (Tube B)

• For each transduction reaction (i.e. one well in a 384-well plate), prepare the transduction solution by mixing 6 μL of the Sensor BacMam stock with 0.3 μL of the 500 mM stock solution of sodium butyrate,  and 18.7 μL of the complete culture media for your cells, for a total volume of 25 μL. Mix gently.

Step 3) Mix Cells and Transduction Mix from above.

• Mix Tube A and Tube B (50 μL tube A + 25 μL tube B). Mix gently and seed 75 μL of the mix per well on the 384-well plate.

* In step 1, the volume of the cell suspension per well can be reduced from 50uL to 25 uL if desired, for a final plating volume of 50uL per well after the completion of step 3. Make sure that the well is still receiving 12,500 cells.

For the best results in CHO cells on a fluorescent plate reader, we recommend ordering our purified, high titer BacMam stock. We recommend using Valproic Acid as an HDAC inhibitor to boost expression in CHO cells at a final concentration of 5 mM.

To set up the assay in CHO cells, in 96-well format, please follow the general protocol for HEK cells in 96-well plates (above), adjusting cell densities and reagent volumes as follows:

Step 1) Prepare cells (Tube A)

• 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.

*210,000 cells/mL works well for CHO cells. This will result in 21,000 cells/well in a 96-well plate. Depending on the plate type being used, 20,000-22,000 cells per well may be optimal.

Step 2) Prepare Viral Transduction Reaction (Tube B)

• For each transduction reaction (i.e. one well in a 96-well plate), prepare the transduction solution by mixing *5 μL of the Purified Sensor BacMam stock with 2.5 μL of the 300 mM stock solution of Valproic Acid, and 42.5 μL of the complete culture media for your cells, for a total volume of 50 μL. Mix gently. 

*5μL of Purified Sensor BacMam stock is a good starting point, but a range of 2-10 μL may need to be tested if 5μL is suboptimal.

• Optional but recommended: Replace the media with 150 μL of fresh media. The viral transduction mix contains autofluorescence, thus changing the media will increase the signal:noise in the assay. 
• Prepare positive control compound. Add 50 μL of 4 μM thapsigargin diluted in buffer of choice from the 100 μM stock to a set of wells. This creates a 1 μM concentration of thapsigargin in 200 μL of media/buffer. 
• Add desired compounds to each well, return plate to incubator and allow the fluorescence to equilibrate for 1 hour. Reserve a few wells to add thapsigargin, your positive control compound.
• After 1 hour take an initial fluorescent reading or image. Experiments are performed using standard GFP excitation and emission wavelengths.
• Read or image the fluorescence intensity from the plate at reasonable time points after the addition of compounds. Cells treated with thapsigargin will begin to increase in fluorescence as soon as 3 hours after initial treatment and will reach a peak intensity 7 hours after treatment (See Figure 2).
• When monitoring the green fluorescence emitted by the sensor, either the change in fluorescence intensity over time or the absolute fluorescent intensity can be measured. 
• Different compounds induce cellular stress at different rates. Thus, we suggest taking multiple fluorescence measurements over a 24-48 hour period.
• If a single endpoint measurement is required, the media can be exchanged for DPBS after the desired incubation time followed by either image or plate reader analysis.

Fluorescence Detection

Our assays are compatible with automated fluorescent plate readers. Our customers have reported good results on:

• Hamamatsu FDSS
• Molecular Devices FLIPR
• Molecular Devices Flexstation
• Perkin Elmer Enspire
• Biotek Lionheart FX

We have validated on:

• Biotek Synergy MX
• Biotek Cytation
• BMG CLARIOstar
• Epifluorescence microscopes

Fluorescence Properties

The Cell Stress Sensor is constructed with the very bright, mNeon green fluorescent protein [6], and the constitutively expressed red fluorescent protein is SantakaRFP. While the optimal excitation and emission wavelengths for mNeon green are 506 nm and 517 nm, respectively, a range of 485-505 nm for excitation and 515-535 nm for emission may be used. We recommend Chroma’s Catalog set #49003 for optimal results. Preferred excitation and emission wavelengths for mNeon Green are 485/528 and preferred excitation and emission wavelengths for SantakaRFP are 558/603.

Figure 1A. Absorption and emission properties of the mNeon green fluorescent protein plotted as a function of wavelength.

Figure 1B. Absorption and emission properties of a red fluorescent protein plotted as a function of wavelength.

Timing

The cell stress assay is a live cell assay, allowing detection over short and long time intervals. For best results, be sure to analyze fluorescence between 0 and 1 hour after addition of stress inducing compounds and at multiple time points up to 48 hours after to capture changes cellular stress levels.  In Figure 2A, fluorescence was captured from cells at 1 hour after compound addition then sampled at regular intervals. The maximal response for the positive control, thapsigargin is reached at 7 hours after the addition of the drug. 

Figure 2: Monitoring changes in cell stress with the two-color cell stress biosensor. A: HEK293T cells transduced with the two-color cell stress biosensor and treated with increasing amounts of thapsigargin. Green fluorescence was monitored for 24 hours on a BMG CLARIOstar plate reader with the atmosphere control unit, temperature at 37°C and 5% CO₂. Note that stress induction, indicated by an increase in green fluorescence, appears within 60 minutes, and begins to decrease by 7 hours. B: HEK293T cells transduced with the two-color stress sensor and treated with increasing concentrations of thapsigargin. These data were analyzed in the BioTek Lionheart FX high content imaging system. The percentage of stress cells over time was used to quantify cell stress levels. C: EC50 values from A and B comparing analysis of the stress sensor on plate reader or imaging systems. D: Zʹ quantification of the cell stress sensor using either green or green/red fluorescence values. 

The protocol above is optimized for rapidly dividing immortalized cells. However, these assays are compatible with screening primary cultures and iPSC-derived lines, where the cells are plated before transduction. Specific details of the protocol will vary by cell type, so it is important to take the time to titrate BacMam for optimal results.  For expression in rare cell types, or specific cells in mixed cultures, Cre-dependent and specific promoter systems are available for many of our sensors.

• Prepare a 500 mM stock solution of sodium butyrate in sterile water (provided in your kit).
• For each transduction reaction (i.e. one well in a 96-well plate, containing 100μL culture media per well), prepare a transduction solution by mixing 20 μL of the Sensor BacMam stock with 5 μL of Receptor control, 24.4 μL of DPBS, and 0.6 μL of the 500 mM stock solution of sodium butyrate for a total volume of 50 μL. Mix the solution gently.
• Sensor expression and cell health can be controlled by titrating the virus, so it is worth taking the time to optimize the assay for your particular cell type. Cell Culture media may be used in place of DPBS in the step above. See the assay optimization section for more information.
• Add the transduction reaction directly to the plated cells (no aspiration of cell medium necessary). Gently rock the plate 4-5 times in each direction to mix throughout the well. Incubate the cells under normal growth conditions (5% CO₂and 37°C), protected from light, for 20-24 hrs.
• Optional step (cell type dependent): After 4-8 hr incubation with Sensor BacMam (6 hrs is optimal), aspirate transduction solution and add 100 μL complete growth medium with sodium butyrate at a concentration of 2 mM. Return cells to normal growth conditions for approximately 16-20 hrs before measuring fluorescence as described above. If cells will not tolerate a full media exchange, partial media exchanges can be done.

Are your cells fluorescent?

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 may be important to maintain expression of the sensors. While BacMam transduction alone will result in sensor expression, sodium butyrate or another HDAC inhibitor, such as valproic acid (VPA) or trichostatin A (TSA), will generate higher levels of expression and will maintain this level of expression [Kost, T. et. al. 2007]. If cells look unhealthy, use lower concentrations or no HDAC inhibitor. 

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.

Optimizing expression of the fluorescent sensor

It is important to optimize expression for your particular cell type. Too little virus will produce variable results, particularly if the sensor expression levels are low and difficult to detect on your instrument. Baseline fluorescence goes up as you add more virus, and when a particular threshold is reached the absolute change in sensor fluorescence and the Z’ for the assay become constant.

To determine optimal conditions for your cell type, prepare a dilution series of transduction reactions by varying the amount of BacMam. For example, a range of 5 μL to 50 μL is a good starting range.

Varying the cell density, concentration of sodium butyrate, or trying a new HDAC inhibitor (VPA or TSA) may boost expression as well.

Please contact us if you would like to use the sensor under the control of a specific promoter system. Sensors under weak promoters may be limited to detection on imaging systems. To maintain strong expression in specific cell types, we recommend ordering a Cre-inducible, floxed sensor.

We also provide a more highly refined and concentrated viral preparation upon request, which can increase expression in particularly sensitive or difficult to transduce cell types.

Is the positive control working?

In cells that are expressing the sensor, addition of the positive control compound, thapsigargin, will result in increased green fluorescence within 7 hours. Both the green sensor and constituitively active red label are localized to the nucleus to simplify segmentation for high content imaging applications.

A serial dilution series of the sensor with a constant amount of thapsigargin positive control can be used to find the optimal expression for your cell type. It is important to determine the kinetics of the response and to set your instrument to measure in the appropriate time frame while avoiding oversampling.

The two-color cell stress biosensor is a useful tool for determining ER-mediated cell stress on both image-based and plate reader-based systems. Discussed here are important aspects of the two-color stress sensor to be considered when analyzing data generated using this biosensor.

Changes in protein expression 

Expression of mutant genes or treatment of cells with exogenous compounds can alter protein expression. Moreover, activation of cell stress can also alter protein expression. The constitutively expressed, nuclear localized red fluorescent protein can serve as an indicator of changes in overall protein expression. The green stress-induced fluorescence can be compared to the red fluorescence to normalize for changes in overall protein expression. However, as many compounds or mutations can alter cellular expression levels over different time frames it is important to explore changes in protein expression and cell stress activation over a range of time frames.

Plate reader analysis

When analyzing the cell stress sensor on a standard fluorescence plate reader either green fluorescence intensity or green/red ratio fluorescence can be used to determine cellular stress. (Figure 2D)

Image Analysis

When using an imaging-based approach to detect cell stress the same fluorescence intensity measurements of green or green/red ratio fluorescence can be used. However, another method that can be used with imaging and high content based analysis is the percent of stressed cells in the region of interest.In this readout, all cells within a region of interest expressing the biosensor are marked in red, while only the stressed cells within the region express green fluorescence. Dividing the number of cells expressing both green and red fluorescence by all cells expressing red fluorescence gives the fraction of stressed cells within the imaged region. This type of measurement does not rely on fluorescent intensities that may be altered by overall protein expression changes and may be useful when analyzing a number of different stress inducing compounds that alter general protein expression. Importantly, both intensity based and percentage based quantifications of cell stress produce highly similar results as shown in Figures 2 and 3.

Figure 2: Monitoring changes in cell stress with the two-color cell stress biosensor. A: HEK293T cells transduced with the two-color cell stress biosensor and treated with increasing amounts of thapsigargin. Green fluorescence was monitored for 24 hours on a BMG CLARIOstar plate reader with the atmosphere control unit, temperature at 37°C and 5% CO₂. Note that stress induction, indicated by an increase in green fluorescence, appears within 60 minutes, and begins to decrease by 7 hours. B: HEK293T cells transduced with the two-color stress sensor and treated with increasing concentrations of thapsigargin. These data were analyzed in the BioTek Lionheart FX high content imaging system. The percentage of stress cells over time was used to quantify cell stress levels. C: EC50 values from A and B comparing analysis of the stress sensor on plate reader or imaging systems. D: Zʹ quantification of the cell stress sensor using either green or green/red fluorescence values. 

Figure 3. Plate reader and image based analysis of the two-color cell stress biosensor. Left: EC50 values determined in HEK293T cells for the ER stress inducing compound thapsigargin using either green fluorescence intensity from plate reader-based measurements (left) or percent stressed cells within a region of interest of an image (right). Note that the EC50 values determined for thapsigargin are highly similar between the plate reader and image based analysis. Data are plotted as mean ± SD, n=3 wells. Right: Images of HEK293T cells treated with different concentrations of thapsigargin. Note the dose dependent increase in the percent of cells expressing the green stress marker in each image.

Storage

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

BioSafety Considerations

The BacMam vector carrying the fluorescent biosensors used in these assays is a modified baculovirus, which can be used for delivery to, and expression in, a wide variety of mammalian cell types including primary cultures.

BacMam is a modified baculovirus, Autographa californica, AcMNPV. The natural host of baculovirus is larvae of the order Lepidoptera.The BacMam vector in the kit is produced in the lab using Sf9 insect cells and 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.

Other types of viruses are quantified in terms of plaque forming units (PFU) in cells from the natural host. Since BacMam is modified to produce expression in mammalian cells, we quantify the virus by measuring viral genes (VG) per milliliter (mL). Viral samples are prepared to release viral genomic DNA, then multiple dilutions of the preparation are run in qPCR using primers that are specific to the VSVG gene in the BacMam genome. Results are compared 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 stock.

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.

Problem 1: Low sensor expression and/or poor transduction efficiency

Problem 2: Low fluorescence signal on microscope/plate reader

Problem 3: Signal-to-background is low (i.e. cells/wells with sensor are not much brighter than control cells/wells without sensor)

Problem 4: Signal is noisy

Problem 5: Good fluorescence signal, but sensor is not responding to drug as expected. No change in fluorescence observed, or signal is in the wrong direction.

Problem 6: Poor cell health, cells detaching from plate.

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Problem 1: Low sensor expression and/or poor transduction efficiency

• Possible Cause—Suboptimal sensor BacMam volume is being used

Solution→ Perform titration of the sensor BacMam stock, testing a large range (i.e. 5-50μL in 96-well plate format) to identify optimal volume. Too little can result in low expression, too much can cause cells to become sick.

 

• Possible Cause—Transducing adherent cells

Solution→ Transduce cells while in suspension. If this isn’t possible, try doing a media exchange on adherent cells after 4-6hrs, in addition to leaving the virus on overnight.

 

• Possible Cause—Sub-optimal cell density; too few or too many cells added

Solution→ Transduce cells so that the cells will be around 75-80% confluent at the time of transduction. Also, when transducing cells in suspension, make sure that cells in the source flask are < 100% confluent (approximately 80% confluent is ideal).

 

• Possible Cause—HDAC inhibitor was not added to the transduction mix, or the concentration was wrong

Solution→ Add HDAC inhibitor at the proper concentration:

a) Sodium Butyrate – 2mM

b) Valproic Acid – 5mM

c) Trichostatin A – 0.25μM

*A titration can be performed to determine optimal concentration for the cell type being used.

 

• Possible Cause—HDAC inhibitor being used is not optimal for cell type

Solution→ Test other HDAC inhibitors (e.g. Sodium Butyrate, Valproic Acid, Trichostatin A.)

 

• Possible Cause—Cell type being used transduces poorly

Solution→

a) After adding transduction mix to cells, let cells sit at room temperature for 30-40 min before placing back in incubator (longer incubation times at RT may further improve transduction).

b) Perform media exchange after various incubation times with the transduction mix, in addition to leaving the virus on overnight.

c) Try high titer, purified BacMam stock.

d) Validate assay in a different cell type (e.g. HEK293 cells)

e) Transduce cells multiple times (e.g. on Day 1, and again on Day 2).

f) Incubate cells for 48 hrs post transduction, before performing assay.

g) Consider using a different viral vector, such as lentivirus or AAV.

 

• Possible Cause—Cell culture media is inhibiting transduction

Solution→ Remove media during transduction, preparing the transduction mix in DPBS and adding to cells. Replace transduction mix with media after 2-4 hours.

 

• Possible Cause—BacMam stock was not stored properly (i.e. not stored at 4℃, exposed to light for long periods, or subjected to multiple freeze-thaw cycles), or the shelf life has been exceeded.

Solution→ Follow guidelines for product storage. BacMam stocks are stable for at least 12 months when stored properly. After this time period, the stock should be reevaluated and compared to previous experiments. Purified BacMam stocks have a shelf life of 3 months.

 

• Possible Cause—BacMam stock was not mixed adequately before transducing cells.

  Solution→ Mix BacMam stock thoroughly before transduction, especially after being stored for long periods.

   

• Possible Cause—Promoter is not optimal for cell type being used.

  Solution→ Identify promoters that work best in the cell type being used. If promoter is not on product list, consult Montana Molecular for custom production services.

 

• Possible Cause—Cells are contaminated

  Solution→ Monitor cells for bacteria, fungi, etc.

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Problem 2: Low Fluorescence Signal on Microscope/Plate Reader

• Possible Cause—Low sensor expression, low transduction efficiency

Solution→ See solutions for Problem 1 above.

 

• Possible Cause—Excitation/emission settings are not optimal for the sensor being used.

Solution→ Refer to protocol for the fluorescence spectra of the sensor. Make sure that filter sets or monochromotors are aligned with the peak excitation and emission wavelengths of the sensor.

 

• Possible Cause—Cells are in cell culture media, and the  media is producing a large fluorescent signal (autofluorescence).

Solution→ Exchange media so that cells are in DPBS at the time of experiment. If cell culture media must be used, try using FluoroBrite media.

 

• Possible Cause—Wrong microplate type is being used.

Solution→ Use black, clear-bottom microplates with low autofluorescence.

 

• Possible Cause—Exposure time or gain setting on instrument is sub-optimal.

Solution→ Test different exposure and gain settings, monitoring how the signal-to-background, and noise in the measurement, changes. Too high can result in saturation and/or photobleaching; too low can result in noisy data.

 

• Possible Cause—Cells were dislodged during media exchange/plate washing.

Solution→ Make sure that media exchange or plate washing is done gently and does not dislodge cells. Confirm with visual inspection on a microscope.

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Problem 3: Signal-to-background is low (i.e. cells/wells with sensor are not much brighter than control cells/wells without sensor)

• Possible Cause—Low sensor expression, low transduction efficiency.

Solution→ See solutions for Problem 1 above.

 

• Possible Cause—Excitation/emission settings are not optimal for the sensor being used.

Solution→ Refer to protocol for the fluorescence spectra of the sensor. Make sure that filter sets or monochromators are aligned with the peak excitation and emission wavelengths of the sensor.

 

• Possible Cause—Exposure time or gain setting on instrument is sub-optimal.

Solution→ Test different exposure and gain settings, monitoring how the signal-to-background, and noise in the measurement, changes. Too high can result in saturation and/or photobleaching; too low can result in noisy data.

 

• Possible Cause—Media exchange was not performed before running the assay; cells are in media rather than DPBS. Cell culture media being used has high autofluorescence.

Solution→ Perform media exchange so that cells are in DPBS at the time of experiment. If cell culture media must be used, try using FluoroBrite media.

 

• Possible Cause—Cells were dislodged during media exchange/plate washing.

Solution→ Make sure that media exchange or plate washing is done gently and does not dislodge cells. Confirm with visual inspection on a microscope.

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Problem 4: Signal is Noisy

• Possible Cause—Low sensor expression, low transduction efficiency.

Solution→ See solutions for Problem 1 above.

 

• Possible Cause—Gain setting or exposure time on instrument is too low.

Solution→ Increase gain setting or exposure time.

 

• Possible Cause—Media exchange was not performed, or plate washing was inadequate, causing high well-to-well variability. Cells are not in DPBS at the time of experiment.

Solution→ Exchange media so that cells are in DPBS at the time of experiment. If cell culture media must be used, try using FluoroBrite media. Make sure that plate washing is highly consistent from well to well.

 

• Possible Cause—Cells were dislodged during media exchange/plate washing.

Solution→ Make sure that media exchange or plate washing is done gently and does not dislodge cells. Confirm with visual inspection on a microscope.

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Problem 5: Good fluorescence signal, but sensor is not responding to drug as expected. No change in fluorescence observed, or signal is in the wrong direction.

• Possible Cause—Photobleaching

Solution→ Reduce exposure time, sampling rate, and/or light intensity.

 

• Possible Cause—Drug is at the wrong concentration.

Solution→ Confirm drug concentration and solubility.

 

• Possible Cause—Drug was not stored properly.

Solution→ Confirm drug storage conditions.

 

• Possible Cause—Drug was added to the cells in a volume that was too low relative to the volume of DPBS/Media in the well, resulting in improper mixing.

Solution→ Add drug in a volume that will allow for sufficient diffusion (i.e. 1:3 or 1:4 drug to total volume)

 

• Possible Cause—Drug was not added in the same solution as the solution in the well/culture dish.

Solution→ Make sure that the drug preparation and cells are in the same solution.

 

• Possible Cause—Drug addition is producing an artifact.

Solution→ Make sure to add a vehicle-only control. Make sure drug is added in a solution that is the same as the solution in the well. Do not exceed 1% DMSO final in the well (0.5% or less is ideal).

 

• Possible Cause—Compounds being tested are fluorescent.

Solution→ Scan compounds for fluorescence to confirm. If possible, dilute compounds in order to reduce the fluorescence artifact of the compound.

 

• Possible Cause—Drug addition was too forceful and dislodged cells.

Solution→ Add drugs manually or with an on-board dispense function, but do so gently, so as not to dislodge cells.

 

• Possible Cause—Gain setting on instrument is too high, and signal is saturating. Gain setting is too low, and signal cannot be detected.

Solution→ Adjust gain setting.

 

• Possible Cause—Too much sensor has been added to cells and the signal is saturated (i.e. not enough analyte for the amount of sensor in the cell).

Solution→ Titrate the amount of sensor to determine maximum signal for your cell type. See protocol for recommendations for HEK293 and CHO cells.

 

• Possible Cause—Target receptor was not added, or expression levels are suboptimal (too little or too much, or receptor has high level of constitutive activity).

Solution→ Titrate the amount of receptor to optimize the signal for your cell type and receptor.

 

• Possible Cause—Sampling rate is not consistent with sensor kinetics.

Solution→ Acquire 5-10 baseline measurements before adding drug. Resume measurement quickly after adding drug (within 5-10 seconds for DAG/PIP₂, 60 seconds for cADDis and cGMP, and 1-2 seconds for GECO). Measure long enough to capture max response of sensor.

 

• Possible Cause—Baseline reads were not acquired before adding drug.

Solution→ Acquire 5-10 baseline fluorescence reads before adding drug. Monitor for a change in fluorescence intensity upon addition of drug.

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Problem 6: Poor cell health, cells detaching from plate.

• Possible Cause—Too much BacMam stock was added to cells (e.g. sensor, receptor, Gs mutant).

Solution→ Titrate lower amounts of BacMam stock to identify the optimal volume for your cells.

 

• Possible Cause—Concentration of HDAC inhibitor is too high, or cells are sensitive to the HDAC inhibitor being used.

Solution→ Confirm concentration of HDAC inhibitor being used. Make new stock solution. Try a different HDAC inhibitor. Confirm that they are being used at the proper concentration:

a) Sodium Butyrate – 2mM

b) Valproic Acid – 5mM

c) Trichostatin A – 0.25μM

*A titration can be performed to determine optimal concentration for the cell type being used.

 

• Possible Cause—Plate surface is not coated with a cell attachment factor.

Solution→ Coat plates with a cell attachment factor (e.g. PDL, laminin, collagen, fibronectin etc.) to enhance attachment

    .

• Possible Cause—Edge wells are being used, and cells in the edge wells may be subject to conditions that are not conducive to growth.

Solution→ Do not use edge wells.

 

• Possible Cause—Cells were harmed or dislodged during media exchange/plate washing.

Solution→ Make sure that media exchange or plate washing is done gently and does not dislodge cells. Confirm with visual inspection on a microscope.

 

• Possible Cause—DPBS being used does not contain calcium and magnesium.

Solution→ Use DPBS containing calcium and magnesium.

 

• Possible Cause—Cells are contaminated.

Solution→ Monitor cells for bacteria, fungi, mycoplasma.

 

• Possible Cause—Cells were not grown under proper growth conditions (i.e. 5% CO₂, 37°C).

Solution→ Incubate transduced cells at 37°C, in 5% CO₂.

If you have any questions about the protocols described here, or if you have ideas about how we can improve these tools, then we want to hear from you. Your feedback is extremely valuable. Please send an email to [email protected], and we’ll respond as quickly as we can.