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.

-Cell stress sensor BacMam ⩰5 x10*10VG/mL in ESF 921 Insect Cell Culture Medium (Expression Systems #96-001-01).
Green fluorescent sensors that increase in fluorescence intensity in response to ER and cellular stress. VG/mL is the number of viral genes per milliliter.
-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 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

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. 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 or FluoroBrite media and determine cell count. Note: The assay will work in either standard media or FluorBrite media, however as FluoroBrite media is formulated to reduce background fluorescence signals and signal to noise ratios are generally improved using FluoroBrite media. Alternatively, if only a single time point the media can be exchanged for DPBS before analysis.
• 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.

*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 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. This step is important to maintain even distribution of cells throughout the well and minimize edge effects.
• 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

• 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 for 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. 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
The fluorescent signal is produced by the mNeon green fluorescent protein [6]. We recommend Chroma’s Catalog set #49003 for optimal results. See the spectrum in Figure 1 on the next page.

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 2, 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

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 our goal for this assay, we quantify infectivity by measuring viral genes (VG) per milliliter (mL) of the BacMam stock. Viral samples are prepared to release 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 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.

The protocol above is optimized for rapidly dividing immortalized cells. However, the cell stress 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.

• Prepare a 500mM stock solution of sodium butyrate in sterile water.
• 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 25μL of the cell stress sensor BacMam stock with 24.4μL of DPBS, and 0.6μL of the 500mM stock solution of sodium butyrate for a total volume of 50μL. Mix the solution gently.
• 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.
• Prepare a dilution series of transduction reactions by varying the amount of BacMam. For example, a range of 10μL to 50μL, adjusting the amount of DPBS accordingly.
• Add the transduction reaction directly to 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% CO2 and 37°C), protected from light, for at least 4 hours, overnight is preferable.
• Aspirate transduction solution and add 100μL complete growth medium with sodium butyrate at a concentration of 3-6mM. Return cells to normal growth conditions for approximately 18-22 hours 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?

Different types of promoters drive expression in mammalian cells. The CMV promoter is effective in many cell lines, but not all. If you have a particular promoter you know works best in your cells, let us know. 

Twenty four hours after transduction, you should see bright green fluorescent cells with an epifluorescence microscope. If using a plate reader, reserve a few control wells with untransduced cells. The transduced wells on the plate should be significantly more fluorescent than untransduced cells on the same plate.

An HDAC inhibitor may be used to boost expression of the sensors.  While BacMam transduction alone will produce expression, sodium butyrate or another HDAC inhibitor such as valproic acid or trichostatin A (TSA) may help maintain the level of expression over time [Kost, T. et. al. 2007]. If cells look unhealthy, use lower concentrations or none of the 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. 

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. Fluorescence from the green stress sensor alone (#U0900G) is localized throughout the cell. 

Addition of thapsigargin results in an increase in green fluorescence, as shown in Figure 2.  If it does not, then it is important to use the positive control to optimize the amount of sensor, the HDAC inhibitor concentration, or compound concentrations. A serial dilution series of the sensor with a constant amount of thapsigargin positive control can be used to find the optimal expression. 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.

Figure 2.  HEK 293 cells transduced with 25 μL of green cell stress sensor and activated with either thapsigargin or tunicamycin. The plot displays the percent change in fluorescence over time compared to 1 hour after addition of either drug or DMSO control. Data points are plotted as the mean ± standard deviation from 6 wells for each sample on a 96-well plate. The Zʹ for thapsigargin after 8 hours is 0.74.