Red PIP2 Assay Protocol

About This Assay #D0400R Red PIP2 Sensor

PIP2, also known as Phosphatidylinositol 4,5-bisphosphate or PtdIns(4,5)P2, is a substrate for several signaling molecules. It is a precursor for the DAG/IP3 pathway, but also plays a key role in phospholipid signaling and is involved in the regulation of ion channels and transporters. The fluorescent sensors used in the assays described here can be combined with different colored sensors, such as green calcium sensors, or a green DAG sensor, to measure multiple signals simultaneously.

Overview

The red PIP2 sensor is based on a dimerization-dependent fluorescent protein. This protocol is optimized for imaging rapidly dividing, immortalized cell lines on a 96-well plate and has been validated in live HEK293, CHO and NIH 3T3 cells. For imaging live iPSC-derived or primary cultured cells, see Suggestions for 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.

Materials in the Kit

PIP2 sensor BacMam in TNM-FH Insect Culture Medium (Allele Biotech product #ABP-MED-10001).
-Red fluorescent sensors that decrease in fluorescence intensity when PIP2 levels decrease. VG/mL is the titer determined by qPCR, and is the average number of viral genes per mL of the BacMam stock.

Sodium Butyrate (Sigma Aldrich product number B5887) 500 mM in H2O.
-Sodium Butyrate maintains BacMam expression in HEK293 cells.  Other HDAC inhibitors such as Trichostatin A (TSA) or Valproic acid (VPA) may be substituted.

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, provided as a positive control for the purpose of assay optimization. Separately expresses a green fluorescent protein that is targeted to the nucleus.

Carbachol 25 mM in H2O
-Carbachol can be used to stimulate Gq signaling through the positive control, the M1 muscarinic acetylcholine receptor.

 

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].

Storage/Biosafety/Warranty

Baculovirus stock should be stored at 4°C and protected from light. Avoid freeze/thaw cycles.

BioSafety Considerations
BacMam does not replicate in mammalian cells and expresses only the fluorescent sensor.  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.

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. 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.

*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 20-25 μL of the BacMam stock with 0.6 μL of the 500 mM stock solution of sodium butyrate**, 5 μL of receptor control, and enough complete culture media 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 uL Receptor Control x 110 wells = 550uL
0.6 μL 500 mM Sodium Butyrate x 110 wells = 66 μL
24.4 μL 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 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. 
  • Incubate for ≈ 44-48 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 Incubation

  • (Optional) A partial media exchange during this incubation period may improve cell health. With approximately 150 μL total volume in the well, remove 75 μL and add 75 μL of fresh culture media, making sure to maintain the concentration of sodium butyrate.
DAY 3: Measuring Fluorescence

Day 2: Incubation

  • (Optional) A partial media exchange during this incubation period may improve cell health. With approximately 150 μL total volume in the well, remove 75 μL and add 75 μL of fresh culture media, making sure to maintain the concentration of sodium butyrate.

Day 3: MEASURING FLUORESCENCE

  • Cells are now ready for assay. Prior to imaging, 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 using standard RFP excitation and emission wavelengths. 
  • Add 50μL of 200μM carbachol (50 μM final concentration per well) to activate the DAG/IP3 pathway in a set of control wells.  A decrease in the fluorescence intensity will be observed after addition of the carbachol when PIP2 is hydrolyzed to produce IP3 within the cell.

Fluorescence Detection

This sensor is constructed using a red fluorescent protein. Use 572/20 nm excitation and 630/30 nm emission filters to collect the red fluorescence from the red PIP2 sensor. These filter properties are similar to many of the FITC filter sets commonly available on most microscopes.

 

 

 

 

 

Timing

The PIP2 assay measures PIP2 in living cells, in real time.  Be sure to capture fluorescence during the peak response as shown in Figure 2, which provides an example experiment using the Green PIP2  sensor.

Figure 2. Green fluorescent PIP2 sensor in HEK 293 cells, co-transduced with human muscarinic acetylcholine control receptor M1, and a red nuclear label.  Fluorescence was captured from cells before the addition of 50 μM carbachol, and sampled at regular intervals. The maximal response is reached at ~60 seconds after the addition of the drug, and the response begins its return to baseline ~100 seconds after drug is added.

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 (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 PIP2 sensor stock.

Level of sensor expression:
To optimize the assay in your particular cell type, it is important to optimize the amount of BacMam 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.

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 receptors can artificially change the resting levels of second messengers.

 

Suggestions for Assays in Adherent Cells

The protocol above is optimized for rapidly dividing immortalized cells, however, this product is compatible with screening primary cultures and iPSC-derived lines, where the cells are plated before transduction. Specific protocols will vary by cell type, so it is important to take the time to titrate the BacMam stock for best 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.
  • For each transduction reaction (e.g. one well in a 96-well plate, 100 μL per well), prepare the  transduction solution by mixing 25 μL of the BacMam stock with 23.5 μL of DPBS and 1.5 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.
  • Prepare a dilution series of transduction reactions by varying the amount of BacMam ranging from 15 μL to 80 μL and adjusting the amount of DPBS accordingly.
  • 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, protected from light, for 6 hours (5% CO2  and 37°C).
  • Aspirate transduction solution and add 100 μL complete growth medium with sodium butyrate at a concentration of 1-2 mM. Return cells to normal growth conditions for approximately 24 hrs.
Troubleshooting

If you encounter problems, there are several steps that you can consider when 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.  If your cells are not fluorescent, check that the CMV promoter works in your cell line.  40-48 hours after transduction with the downward PIP2 sensor, 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 maintain expression of the sensors.  While BacMam transduction without the HDAC inhibitor will initially generate low levels of sensor expression, it is important to follow protocols and include sodium butyrate or another HDAC inhibitor such as VPA, or trichostatin A (TSA) to generate optimal levels of sensor expression.

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 receptor included in this kit.  Adding 5 μL of the hM1 receptor stock to set of control wells will ensure that a Gq coupled receptor is present in all of the cells.  You can double check to make sure the M1 receptor is expressed by examining the cells in a fluorescent microscope with filters for green fluorescence.  You should see green nuclear fluorescence that marks the cells that express the exogenous M1 receptor.

Addition of carbachol will cause a change in fluorescence, when the receptor control is present in the cells, as shown in Figure 2.  This positive control can be used to optimize three aspects of your assay. First, a serial dilution series of the sensor with a constant amount of PIP2 sensor can be used to determine the optimal sensor expression for your instrument and cell type.  Second, it is important to titrate the amount of BacMam sufficient to transduce all of the cells in the well.  Third, it is important to determine whether your instrument can measure the peak response in the appropriate time frame.

Contact Us

If you have any questions about the protocols described here, or if you have ideas about how we can improve these tools, then we hope 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.

References

1. Graham FL, Smiley J, Russell 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.

2. Dulbecco R and Vogt M: Plaque formation and isolation of pure lines with poliomyelitis viruses. The Journal of experimental medicine 1954.

3. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 1994.

4. 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.

5. Tewson P, Quinn AM, Hughes TE:. A Multiplexed Fluorescent Assay for Independent Second-Messenger Systems: Decoding GPCR Activation in Living Cells. Journal of Biomolecular Screening 2013 vol. 18 no. 7 797-806.