Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The JAK/STAT signaling pathway plays a key role in mediating cellular responses to diverse cytokines, interferons, growth factors, and related molecules^1,2. The binding of these ligands to specific cell-surface receptors results in the activation of JAKs, which in turn activate STAT proteins by phosphorylation of specific tyrosine residues. STAT phosphorylation results in their dimerization and translocation into the nucleus, where they exert their effect on the transcription of regulated target genes. The STAT family consists of seven members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. The members play a complex and essential role in the regulation of physiologic cell processes, including proliferation, differentiation, apoptosis, angiogenesis, and immune system regulation. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions^3,4. Therefore, the ability to assess STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research.

To date, the conventional methods used to measure intracellular phosphorylated protein levels, including STATs, are antibody-based and include western blotting, ELISA, and phosphoflow cytometry. These heterogeneous methods are labor-intensive, time-consuming, error-prone, low-throughput, and often unreliable (e.g., specificity issues) in the case of western blotting⁵. In contrast, homogeneous assays require fewer experimental steps, use smaller sample volumes, and are amenable to HTS. There are five homogeneous cell-based immunoassay platforms commercially available that can be used to quantitatively monitor JAK-dependent phosphorylation of STATs in cell lysates: SureFire, HTRF, LANCE, LanthaScreen, and Lumit. Each of these platforms has its advantages and disadvantages.

SureFire is based on luminescent oxygen channeling technology, which utilizes donor and acceptor beads coated to specifically capture a pair of antibodies, one of which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the donor and acceptor beads into close proximity, enabling the generation of a chemiluminescent signal⁶. While versatile and sensitive, this technology is expensive, is affected by biotin in the culture medium, is very sensitive to ambient temperature and light, and requires a special reader for detection. HTRF and LANCE are both based on TR-FRET technology that utilizes long-lifetime luminescent lanthanide ion complexes (Europium or Terbium chelates, or Europium cryptate) as the donor molecules and far-red fluorophores as the acceptor molecules⁷. When two protein-specific antibodies labeled with either donor or acceptor molecules are brought into close proximity, FRET takes place, causing an increase in acceptor fluorescence and a decrease in donor fluorescence. These long-lived fluorescent signals can be measured in a time-resolved and ratiometric manner to reduce assay interference and increase data quality. Other advantages of TR-FRET are that it is not light-sensitive, allows repeated readings, and exhibits long signal stability. While TR-FRET is widely implemented in HTS due to its versatility, sensitivity, and high robustness, all commercial TR-FRET-based assay platforms are expensive, thereby precluding its wide adoption in academic and small industrial laboratories. The LanthaScreen assay also uses a TR-FRET based-readout but is reliant on an engineered U2OS cell line that stably expresses green fluorescent protein (GFP)-STAT1 fusion protein combined with a terbium-labeled phospho-specific STAT1 antibody⁸. In addition to being limited in terms of choice of signaling proteins, this method requires purchasing expensive transfected cell lines, reducing its applicability and increasing the possibility of experimental artifacts. Lumit is a generic bioluminescent immunoassay platform that utilizes secondary antibodies (anti-mouse and anti-rabbit) chemically labeled with the small and large NanoBit subunits of NanoLuc Luciferase⁹. The binding of two primary antibodies to the target protein brings the secondary antibodies into proximity to form an active enzyme that generates a luminescence signal. While luminescence is generally a sensitive and robust readout, the requirement for primary antibodies raised in two different species limits the choices for assay design. In addition, the use of secondary antibodies in complex sample matrixes may be prone to assay interference.

Thus, a need still exists for a reliable, rapid, yet affordable cell-based assay platform for measuring individual phosphorylated and total STAT proteins in a manner compatible with HTS. To address this need, a new high-throughput cell-based immunoassay platform was developed based on an enhanced TR-FRET technology (THUNDER) and designed to enable simple, sensitive, robust, and cost-effective measurement of endogenously expressed intracellular proteins (phosphorylated or total) in cell lysates. The advantages of this technology stem from the combination of a donor/acceptor FRET pair exhibiting exceptional spectral compatibility and TR-FRET signal, rigorously validated antibodies, and optimized lysis buffers. These assays are formatted as sandwich immunoassays and use a straightforward, three-step workflow (Figure 1). Cells are first treated to modulate protein phosphorylation and then lysed with the specific lysis buffer provided in the kit. The target phosphorylated or total STAT protein in the cell lysate is detected in a single reagent addition and incubation step with a pair of fluorophore-labeled antibodies that recognize distinct epitopes on the target protein (Figure 2). One antibody is labeled with a Europium chelate donor (Eu-Ab1), while the second antibody is labeled with a far-red acceptor fluorophore (FR-Ab2). The two labeled antibodies bind to the protein in solution, bringing the two labels into close proximity. Excitation of the donor Europium chelate at 320 or 340 nm triggers a FRET to the acceptor, which emits a long-lived TR-FRET signal at 665 nm proportional to the concentration of target protein (phosphorylated or total) in the cell lysate.

Figure 1: TR-FRET assay workflow. The workflow consists of three steps: cell treatment, cell lysis, and protein detection using TR-FRET. In the two-plate assay protocol, lysates are transferred to a white 384-well detection plate, whereas in the one-plate protocol, all steps are conducted in the same white 384-well detection plate (all-in-one-well protocol). Regardless of the assay protocol used, protein detection is performed in the same total volume (20 µL per well). Abbreviation: TR-FRET = time-resolved Förster resonance energy transfer. Please click here to view a larger version of this figure.

Figure 2: TR-FRET sandwich immunoassay principle. One antibody is labeled with the Europium chelate donor (Eu-Ab1) and the second with the far-red small fluorophore acceptor (FR-Ab2). The two labeled antibodies bind specifically to distinct epitopes on the target protein (phosphorylated or total) in the cell lysate, bringing the two fluorophores into close proximity. Excitation of the donor Europium chelate at 320 or 340 nm triggers a FRET from the donor to the acceptor molecules, which in turn emit a signal at 665 nm. This signal is proportional to the concentration of protein in the cell lysate. In the absence of the specific target protein, the donor and acceptor fluorophores are too distant from each other for FRET to occur. Abbreviations: FRET = Förster resonance energy transfer; TR-FRET = time-resolved FRET; Ab = antibody; FR = far-red; Eu - Europium chelate; P = phosphorylation. Please click here to view a larger version of this figure.

Here, detailed protocols are provided for measuring, in a 384-well format, the intracellular levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641), together with total STAT1, STAT3, STAT5, and STAT6, in cell lysates from adherent or suspension cells using the THUNDER TR-FRET platform. These protocols define steps for cell treatment, lysis, and TR-FRET-based target protein detection using either a two-plate transfer protocol or a one-plate all-in-one-well protocol. These cell-based assays are applied for determining the pharmacological profile of known activators and inhibitors of the JAK/STAT pathway. The robustness and suitability of selected assays for HTS are demonstrated. Lastly, key experiments for assay optimization are discussed, along with recommendations for assay troubleshooting.

1. Cell culture

1. Maintain cells in a humidified 37 °C/5% CO₂ incubator and culture with either DMEM supplemented with 10% fetal bovine serum (FBS) (HeLa and A431 cells) or RPMI supplemented with 15% FBS (U266B1 cells). Culture the cells until they reach 70-80% confluence, then trypsinize them and passage or use them for the assays.
   ​NOTE: Culture media contained phenol red. No serum starvation was conducted for any cell line prior to conducting the assays.

2. Stimulator or inhibitor titration using the two-plate assay protocol with adherent cells

NOTE: This procedure describes how to determine stimulator or inhibitor potencies by generating a concentration-response curve from a dilution series of the test compound.

1. Cell seeding
   1. Dispense 50 µL of cells at the pre-optimized density (40,000 HeLa cells/well for both STAT3 and STAT6; 75,000 A431 cells/well for STAT5) into a 96-well tissue culture-treated plate in the appropriate culture medium. Incubate overnight at 37 °C/5% CO₂.
      ​NOTE: Optimal cell density and culture incubation time need to be determined.
2. Dilutions of test compounds
   1. Prepare intermediate 2x dilution series of test compound(s) by serially diluting compound(s) (half-log interval dilutions) across 12 wells of a polypropylene 96-well plate into serum-free medium.
      NOTE: It is recommended to conduct a 12-point, half-log interval concentration-response curve in at least duplicate for an accurate estimation of the EC₅₀ or IC₅₀.
   2. Alternatively, for hydrophobic, dimethylsulfoxide (DMSO)-soluble test compounds, perform the initial dilutions in 100% DMSO, and then dilute the compound dilution series into serum-free medium.
      ​NOTE: The assay tolerance to DMSO must be established before conducting a test compound titration in DMSO vehicle. It is important to keep equal solvent concentrations between treated and untreated cells. In addition, when testing serial dilutions of compounds, the solvent concentrations should always remain constant across the dilution series.
3. Cell treatment
   1. For cell stimulation, add 50 µL of serum-free medium alone (untreated cells) or containing the stimulator (2x).
   2. Incubate for the pre-optimized time at either room temperature (RT) or 37 °C (interferon (IFN) α2b/20 min at RT for STAT3; epidermal growth factor (EGF)/10 min at RT for STAT5; interleukin (IL)-4/20 min at RT for STAT6). Proceed then to section 2.4.
      NOTE: Optimal incubation temperature needs to be determined.
   3. For cell inhibition, add 25 µL of serum-free medium alone (untreated cells) or containing the inhibitor (4x).
   4. Incubate for the pre-optimized time at either RT or 37 °C (JAK Inhibitor 1/30 min at RT for STAT3 and STAT6; Erlotinib/15 min at RT for STAT5).
   5. Add 25 µL of serum-free medium alone (untreated cells) or containing the stimulator (4x) at its EC₈₀.
   6. Incubate for the pre-optimized time at either RT or 37 °C (same conditions as for step 2.3.2).
4. Cell lysis
   1. Prepare the kit's specific 1x Supplemented Lysis Buffer as indicated by the manufacturer.
      ​NOTE: It is mandatory to supplement the 1x Lysis Buffer with the 100x Phosphatase Inhibitor Cocktail diluted to a final concentration of 1x. The 1x Supplemented Lysis Buffer contains 1 mM sodium fluoride, 2 mM sodium orthovanadate, and 2 mM beta-glycerophosphate. Other phosphatase inhibitors are not required and should be avoided. Lysis buffers and phosphatase inhibitors other than those included in the kit are not recommended as they might contain ingredients that could interfere with the measurement.
   2. Carefully remove and discard the cell culture medium by aspirating the supernatant.
   3. Immediately add 50 µL of 1x Supplemented Lysis Buffer.
      NOTE: Lysis Buffer volume (25-50 µL) may be optimized.
   4. Incubate for 30 min at RT under shaking (orbital plate shaker set at 400 rpm; moderate agitation).
      ​NOTE: Lysis incubation time (30-60 min) may be optimized. Lysates can be used immediately for target protein detection or frozen at -80 °C.
5. TR-FRET detection
   1. Prepare the 4x Antibody Detection Mix in 1x Detection Buffer as indicated by the manufacturer.
   2. In this transfer step, carefully pipette 15 µL of cell lysate from the 96-well culture plate to a well of a white, low-volume 384-well microplate.
   3. Add 15 µL of the Positive Control Lysate and 15 µL of 1x Lysis Buffer (negative control) to separate assay wells.
   4. Add 5 μL of 4x Antibody Detection Mix (either Eu-Ab1/FR-Ab2 for detection of the phospho-protein or Eu-Ab3/FR-Ab4 for detection of the total protein) to each of the assay wells.
   5. Cover the plate with a plate sealer and incubate for 1 h up to overnight at RT, depending on the assay (see the corresponding Technical Data Sheet).
      NOTE: Optimal reading time needs to be optimized for each assay and cell line. The plate can be read several times without a negative effect on the assay performance.
   6. Remove the adhesive plate sealer and read the plate on a TR-FRET compatible microplate reader.
      ​NOTE: Filter-based fluorometers are recommended, though some monochromator instruments can be used. Verify that the appropriate optic module (filters and mirror) for TR-FRET is installed. Use an excitation wavelength of 320 or 340 nm to excite the Europium chelate. Read assays at both 615 nm (or 620 nm) and 665 nm to detect both the emission from the donor Europium and the acceptor fluorophore, respectively. The instrument settings will depend on the particular reader. Data presented here were obtained using lamp-based excitation, 90 µs delay, 300 µs integration time, and 100 flashes per well. The phospho-STAT4 assay, however, was read using laser excitation to generate higher signal-to-background (S/B) ratios.

3. Stimulator or inhibitor titration using the two-plate assay protocol with suspension cells

1. Dilution of test compounds
   1. Prepare intermediate 2x dilution series of test compound(s) as described in steps 2.2.1 and 2.2.2.
2. Cell seeding and treatment
   1. Dispense 20 µL of cells at the pre-optimized density (200,000 U266B1 cells/well for STAT1; 400,000 U266B1 cells/well for STAT4) into a 96-well tissue culture-treated plate in the appropriate culture medium. Directly proceed to cell treatment or incubate 2-4 h at 37 °C, 5% CO₂.
      NOTE: This step needs to be optimized for different cell types.
   2. For cell stimulation, add 20 µL of serum-free medium alone (untreated cells) or containing the stimulator (2x).
   3. Incubate for the pre-optimized time at either RT or 37 °C (IFNα2b/15 min at RT for STAT1; IFNα2b/25 min at 37 °C for STAT4). Proceed then to section 3.3.
      NOTE: Optimal incubation temperature needs to be determined.
   4. For cell inhibition, add 10 µL of serum-free medium alone (untreated cells) or containing the inhibitor (4x).
   5. Incubate for the pre-optimized time at either RT or 37 °C (JAK Inhibitor 1/30 min at RT for STAT1 and STAT4).
   6. Add 10 µL of serum-free medium alone (untreated cells) or containing the stimulator (4x) at its EC₈₀.
   7. Incubate for the pre-optimized time at either RT or 37 °C (same conditions as for step 3.2.3).
3. Cell lysis
   1. Prepare the kit's specific 5x Supplemented Lysis Buffer as indicated by the manufacturer.
      NOTE: It is mandatory to supplement the 5x Lysis Buffer with the 100x Phosphatase Inhibitor Cocktail diluted to a final concentration of 5x. The 5x Supplemented Lysis Buffer contains 5 mM sodium fluoride, 10 mM sodium orthovanadate, and 10 mM beta-glycerophosphate. Other phosphatase inhibitors are not required and should be avoided.
   2. Add 10 µL of 5x Supplemented Lysis Buffer.
   3. Incubate for 30 min at RT under shaking (orbital plate shaker set at 400 rpm; moderate agitation).
      ​NOTE: Lysis incubation time (30-60 min) may be optimized. Lysates can be used immediately for target protein detection or frozen at -80 °C.
4. TR-FRET detection
   1. Following cell lysis, conduct the TR-FRET detection step as described in section 2.5 for the 2-plate assay protocol for adherent cells.

4. Stimulator titration using the one-plate assay protocol with adherent or suspension cells

1. Dilution of test compounds
   1. Prepare intermediate dilution series of test compound(s) at 3x by serially diluting compound(s) (half-log interval dilutions) across 12 wells of a polypropylene 96-well plate into serum-free medium.
2. Cell seeding and treatment
   1. Dispense 8 µL of cells at the pre-optimized density (160,000 U266B1 cells/well for STAT4; 80,000 HeLa cells/well for STAT6), in the appropriate serum-free culture medium, into a white, low-volume 384well assay plate. Directly proceed to cell treatment or incubate 2-4 h at 37 °C, 5% CO₂.
      NOTE: The requirement for a cell culture incubation period before treatment needs to be determined for different cell types.
   2. For cell stimulation, add 4 µL of serum-free medium alone (untreated cells) or containing the stimulator (3x).
   3. Incubate for the pre-optimized time at either room temperature or 37 °C (IFNα2b/25 min at 37°C for STAT4; IL-4/20 min at 37°C for STAT6). Proceed then to section 4.3.
3. Cell lysis
   1. Prepare the kit's specific 5x Supplemented Lysis Buffer as indicated by the manufacturer.
      NOTE: It is mandatory to supplement the 5x Lysis Buffer with the 100x Phosphatase Inhibitor Cocktail diluted to a final concentration of 5x.
   2. Add 3 µL of 5x Supplemented Lysis Buffer.
   3. Incubate for 30 min at RT under shaking (orbital plate shaker at 400 rpm).
      NOTE: Lysis incubation time (30-60 min) may be optimized. Lysates can be used immediately or frozen at 80 °C.
4. TR-FRET detection
   1. Add 15 µL of Positive Control Lysate (undiluted) and 15 µL of 1x Supplemented Lysis Buffer (negative control) to separate assay wells.
   2. Add 5 µL of 4x Antibody Detection Mix (either Eu-Ab1/FR-Ab2 for the phospho-protein or Eu-Ab3-/FR-Ab4 for the total protein) prepared in 1x Detection Buffer to each of the assay wells.
   3. Cover the plate with a plate sealer and incubate for 1 h up to overnight at RT, depending on the assay.
      NOTE: Optimal reading time needs to be optimized for each assay and cell line. The plate can be read several times without a negative effect on the assay performance.
   4. Remove the adhesive plate sealer. Read the plate on a TR-FRET-compatible microplate reader.

5. Data analysis

1. Calculate the TR-FRET ratio for each well using the following formula (1):
   (1)
   NOTE: Because the TR-FRET signal is read in a time-resolved mode, background subtraction is usually not necessary. If background subtraction is conducted, use the cell-free wells containing the 1x Supplemented Lysis Buffer (negative control) for background subtraction. Determine the average TR-FRET ratio from the cell-free wells and then subtract this value from the TR-FRET ratio of each well.
2. For concentration-response curves, analyze the data according to a nonlinear regression using the 4 parameter logistic equation (sigmoidal dose-response curve with variable slope) and a 1/Y² data weighting to generate EC₅₀ or IC₅₀ values.
3. For the Z' factor experiment, analyze the data according to the following formula (2)¹⁰
   (2)
   Where µ and σ are the mean values and standard deviations for the positive control (p; stimulated cells) and negative control (n; untreated cells), respectively.

Each THUNDER TR-FRET assay was pharmacologically validated by treating adherent (HeLa or A431) or suspension cells (U266B1) with JAK/STAT pathway-specific activators or inhibitors and then measuring the levels of specific phosphorylated and total STATs, when applicable. Assays were conducted in 384-well format using the two-plate transfer protocol and pre-optimized assay conditions. Figure 3, Figure 4, Figure 5, Figure 6, and Figure 7 summarize representative concentration-response curves obtained for all STAT assays. Overall, all stimulator and inhibitor concentration-response curves for the phospho-STAT assays showed robust TR-FRET signals, wide dynamic ranges, low inter-well coefficient of variation (typically ≤5%), and acceptable S/B ratios. The EC₅₀ and IC₅₀ values reported here are within the range of expected values.

Treatment of cells with the JAK activators IFNα2b (phospho-STAT1, phospho-STAT3, and phospho-STAT4), IL-4 (phospho-STAT6), and EGF (phospho-STAT5) showed the anticipated concentration-dependent increase in STAT phosphorylation at specific tyrosine residues, while the corresponding total STAT proteins (STAT1, STAT3, STAT5, and STAT6) remained stable (Figure 3, Figure 4, Figure 5, Figure 6, and Figure 7, Panel A). The signal decrease (less than 20%) observed for total STAT5 with increasing STAT5 phosphorylation is expected and is due to steric hindrance, where phosphorylation of STAT5 hinders the binding of one of the two anti-total STAT5 antibodies to its respective antigen. The EC₅₀ values were in the sub-nanomolar range for IFNα2b and IL-4 (0.083 to 0.47 nM) and in the low nanomolar range for EGF (12 nM). These values are in agreement with published data^6,11.

To confirm that the signal induced by the activators was mediated by the activation of endogenous receptors, cells were pretreated with increasing concentrations of either JAK Inhibitor 1 (a pan JAK inhibitor) or Erlotinib (an EGFR tyrosine kinase inhibitor) prior to submaximal stimulation (EC₈₀) with the STAT activators. As anticipated, both JAK Inhibitor 1 and Erlotinib inhibited the corresponding phospho-STAT levels in a concentration-dependent manner, with IC₅₀ values ranging between the low nanomolar and the high nanomolar range for JAK Inhibitor 1 (29-759 nM) and in the low nanomolar range for Erlotinib (10 nM) (Figure 3, Figure 4, Figure 5, Figure 6, and Figure 7, Panel B). The IC₅₀ values are consistent with published data^11,12,13. As was the case for stimulation experiments, the levels of corresponding total STAT were not affected by either treatment. Taken together, these results demonstrate the specificity of each assay for its endogenous target STAT protein (phosphorylated or total) and their capacity to profile activators or inhibitors exhibiting a range of potencies.

Assays in which the whole workflow (cell treatment, lysis, and protein detection) is conducted in a single well of a 384-well plate without a transfer step are more suitable for HTS. Accordingly, the phospho-STAT4 and phospho-STAT6 assays were performed using the one-plate protocol. Representative data obtained under optimized conditions are summarized in Figure 8 and Figure 9. Stimulation of either phosphorylated STAT4 by IFNα2b in a suspension cell line (U266B1; Figure 8) or phosphorylated STAT6 by IL-4 in an adherent cell line (HeLa; Figure 9) was obtained with acceptable S/B ratios and EC₅₀ values consistent with those obtained using the two-plate protocol. These data show that the phospho-STAT assays can be successfully adapted from a two-plate transfer protocol to a one-plate all-in-one-well protocol.

The Z'-factor is commonly used in the HTS community for the evaluation of the suitability and robustness of an assay for HTS¹⁰. To further validate the phospho-STAT assays, preliminary Z'-factor studies were manually conducted using the two-plate protocol. To assess assay stability, plates were read following both a 4-h incubation and an overnight incubation. Results obtained for the phospho-STAT1 and phospho-STAT3 assays, using a suspension cell line (U266B1) or an adherent cell line (HeLa), respectively, are summarized in Figure 10. Calculated Z′-factor values were 0.85 for phospho-STAT1 and 0.79 for phospho-STAT3. Following overnight incubation, the Z'-factor values remained stable (0.83 for phospho-STAT1 and 0.78 for phospho-STAT3). Similar Z'-factor values were obtained for the other phospho-STAT assays (STAT4: 0.79; STAT5: 0.78; STAT6: 0.63). A cell-based assay with a Z'-factor ≥ 0.40 is considered suitable for HTS¹⁵. Accordingly, these results demonstrate the robustness of these phospho-STAT assays for HTS applications.

Figure 3: Detection of phospho-STAT1 (Y701) modulation in U266B1 cells. Cells (200,000 cells/well) seeded in a 96-well culture plate were treated with increasing concentrations of either (A) IFNα2b for 15 min at RT or (B) JAK Inhibitor 1 for 30 min at RT, then with 1 nM (EC₈₀) of IFNα2b for 15 min at RT. Following lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated for 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; JAK = Janus kinase; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio. Please click here to view a larger version of this figure.

Figure 4: Detection of phospho-STAT3 (Y705) and total STAT3 modulation in HeLa cells. Cells (40,000 cells/well) cultured overnight in a 96-well culture plate were treated with increasing concentrations of either (A) IFNα2b for 20 min at RT or (B) JAK Inhibitor 1 for 30 min at RT then with 1.5 nM of IFNα2b for 20 min at RT. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated for 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; JAK = Janus kinase; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio. Please click here to view a larger version of this figure.

Figure 5: Detection of phospho-STAT4 (Y693) modulation in U266B1 cells. Cells (400,000 cells/well) seeded in a 96-well culture plate were treated with increasing concentrations of either (A) IFNα2b for 25 min at 37 °C or (B) JAK Inhibitor 1 for 30 min at RT, then with 1 nM of IFNα2b for 25 min at 37 °C. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated overnight at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; JAK = Janus kinase; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio. Please click here to view a larger version of this figure.

Figure 6: Detection of phospho-STAT5 (Y694/Y699) and total STAT5 modulation in A431 cells. Cells (75,000 cells/well) cultured overnight in a 96-well culture plate were treated with increasing concentrations of either (A) EGF for 10 min at RT or (B) Erlotinib for 15 min at RT, then with 73 nM of EGF for 10 min at RT. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated overnight at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; EGF = epidermal growth factor; RT = room temperature; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio. Please click here to view a larger version of this figure.

Figure 7: Detection of phospho-STAT6 (Y641) and total STAT6 modulation in HeLa cells. Cells (40,000 cells/well) cultured overnight in a 96-well culture plate were treated with increasing concentrations of either (A) IL-4 for 20 min at RT or (B) JAK Inhibitor 1 for 30 min at RT, then with 0.5 nM of IL4 for 20 min at RT. Following media removal and lysis, lysates were transferred to a low-volume 384-well white plate, followed by the addition of the Antibody Detection Mix. The plate was incubated for 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IL = interleukin; JAK = Janus kinase; RT = room temperature; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio. Please click here to view a larger version of this figure.

Figure 8: Detection of phospho-STAT4 (Y693) stimulation in U266B1 cells with the one-plate protocol. Cells (160,000 cells/well) seeded in a low-volume 384-well white plate were immediately treated with increasing concentrations of IFNα2b for 25 min at 37 °C. Following lysis, the Antibody Detection Mix was added directly to the lysate. The plate was incubated overnight at RT and then read on a TR-FRET-compatible reader using a flash lamp or laser excitation. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; RT = room temperature; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio. Please click here to view a larger version of this figure.

Figure 9: Detection of phospho-STAT6 (Y641) stimulation in HeLa cells with the one-plate protocol. Cells (80,000 cells/well) seeded in a low-volume 384-well white plate were immediately treated with increasing concentrations of IL-4 for 20 min at RT. Following lysis, the Antibody Detection Mix was added directly to the lysate. The plate was incubated 4 h at RT and then read on a TR-FRET-compatible reader. Data are shown as the mean of triplicate wells per assay point. Error bars indicate standard deviation. Some error bars are smaller than the symbol size. Abbreviations: STAT = signal transducer and activator of transcription; IL = interleukin; RT = room temperature; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio. Please click here to view a larger version of this figure.

Figure 10: Intraplate variability study of the phospho-STAT1 (Y701) and phospho-STAT3 (Y705) assays. (A) Phospho-STAT1 assay: suspension cells (200,000 U266B1 cells/well) were treated either with 10 nM of IFNα2b for 15 min or serum-free medium alone (low controls). (B) Phospho-STAT3 assay: adherent cells (20,000 HeLa cells/well) were treated either with 5 nM of IFNα2b for 20 min or with serum-free medium alone. For both assays, the TR-FRET signal was read after 4 h of incubation. Abbreviations: STAT = signal transducer and activator of transcription; IFN = interferon; TR-FRET = time-resolved Förster resonance energy transfer; S/B = signal/background ratio; CV = coefficient of variation. Please click here to view a larger version of this figure.

Compared to conventional methods for phosphoprotein analysis such as western blotting and ELISA-based methods, the workflow for a THUNDER TR-FRET cellular assay is simple and fast, uses a low-volume sample (15 µL), is designed for HTS in a 384-well format, and is highly amenable to automation. The assay protocol is flexible and can readily be adapted to both medium- and high-throughput applications. Assays can be run using either a two-plate transfer protocol or a one-384-well plate protocol. In the two-plate transfer protocol, cells are seeded, treated, and lysed in one cell culture plate, and lysates are subsequently transferred to a separate white detection plate (half-area 96-well or 384-well plate) for analysis. In a one-384-well plate protocol, the entire workflow is conducted in the same well, eliminating the need for a liquid transfer step. The assays could easily be adapted to the 1536-well format by reducing the volume proportionally. Regardless of the assay protocol, there are only three reagent solutions to prepare and no washing steps, and the assays can be run following an "addition-only" protocol, requiring minimal hands-on time.

A critical step in performing any cell-based assay is the optimization of cell culture and treatment conditions¹⁶. Both the cell number and treatment conditions require careful optimization before running an assay, as these key parameters often vary for each cell line and each phosphoprotein. The optimization of these parameters allows maximizing the assay window, obtaining an optimum performance with a high S/B ratio and low inter-well coefficient of variation, and ensuring the robustness and reproducibility of the results. Cell number, serum-starvation (when appropriate), and stimulation or inhibition time (at either room temperature or 37 °C) should be optimized for each cell line and target protein. Too high or too low cell numbers can negatively influence the modulation of intracellular signaling pathways. Cell seeding densities of 40,000-80,000 cells/well for adherent cells or 100,000-200,000 cells/well for suspension cells are generally acceptable for most cell lines. Of note, the optimal length of time for stimulation and inhibition can vary widely among cell lines and target proteins, from a few minutes to several hours. As such, a time course study is strongly recommended to determine the optimal stimulation and inhibition incubation times, ideally at both room temperature and 37 °C, as incubation temperature affects the kinetics of target protein stimulation.

Troubleshooting a cell-based assay can be difficult and time-consuming because of the many steps involved in the workflow (cell culture, cell treatment, lysis, and protein detection). These assay kits include a positive control lysate. It is recommended to use these positive control lysates and negative control (1x supplemented Lysis Buffer alone) in every experiment. The use of proper controls facilitates troubleshooting, as problems can then be quickly attributed to either the detection step (incorrect reagent preparation and/or assay execution) or the quality of the lysates used in the experiment. The latter is generally due to the use of suboptimal cellular conditions.

An additional advantage of this platform is the availability of a suite of optimized lysis buffers with different stringencies. This allows generating more or less heterogeneous lysates from partial subcellular fractionation. This is useful because signaling proteins are located in various intracellular compartments, and some, like STAT proteins, shuttle between compartments (e.g., between the cytoplasm and the nucleus). Of note, phosphorylated and total STAT1 use lysis buffer 1 because they cannot be detected well with the lysis buffer 2 used for the other STAT proteins. In contrast, other homogeneous methods rely on a single "universal" lysis buffer, which generates a more complex sample that can create non-specific interactions with the antibodies. However, the use of different lysis buffers may preclude the analysis of multiple signaling proteins from a single lysate sample. Nevertheless, one can still test lysates generated with different lysis buffers simultaneously on a single plate for parallel pathway analysis.

In conclusion, the methods presented here based on this homogeneous TR-FRET cellular immunoassay platform offer a high-throughput alternative for monitoring JAK/STAT signaling via the detection and quantification of endogenous phosphorylated STAT1/3/4/5/6 proteins, together with total STAT1/3/5/6, in cell lysates. These assays provide new tools for a more comprehensive analysis of specific STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathways. Given its simplicity, specificity, sensitivity, reproducibility, and cost-effectiveness, this new assay platform represents an attractive alternative to traditional immunoassays, both in an academic setting and in industrial laboratories for HTS applications.