Time-Resolved Forster Resonance Energy Transfer for Monitoring Protein Phosphorylation in Cells

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 the 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 a serum-free medium.
      NOTE: The assay tolerance to DMSO must be established before conducting a test compound titration in a 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.