ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells

1. Generation of a Plasmid that Expresses the Chimeric Construct

NOTE: The aim is to generate a plasmid that expresses the extracellular domain of the receptor of interest fused to the transmembrane and intracellular domains of the mouse CD3ζ chain (Figure 1A).

1. Retrieve the sequence of the extracellular domain of the receptor of interest including the signal peptide. Obtain DNA material that is expected to express this receptor (e.g., cDNA or a plasmid).
2. Obtain DNA material that expresses the transmembrane and the intracellular domains of the mouse CD3ζ chain (e.g., cDNA or a plasmid).
3. Design the sequence of the fusion protein based on the general structure shown in Figure 1A. Make sure that the whole sequence is within the same codon reading frame. This sequence is used to design appropriate primers and verify the final product.
4. Design two PCR reactions to amplify each of the fragments of the fusion proteins separately (Figure 1B, 1C).
   NOTE: The specific conditions for the PCR reactions (e.g., elongation time and annealing temperature) depend on the nature of the primers, which are designed for a specific receptor.
   1. Amplify the extracellular segment of the receptor.
      1. Design a 5' primer that includes part of the signal peptide of the receptor, a Kozak consensus sequence, and an appropriate restriction site (Figure 1B).
      2. Design a 3' primer that flanks both parts of the fusion protein (Figure 1B). For example, for the initial design, this 3' primer can include the last 20 base pairs of the extracellular segment of the receptor and the first 9 base pairs of the CD3ζ segment.
         NOTE: There is no need to add a restriction site to the 3' primer since this primer will be used to generate the fused sequence, and is not used for ligation.
   2. Amplify the CD3ζ segment.
      1. Design a 5' primer that flanks both parts of the fusion protein (Figure 1C). For example, for the initial design, the 5' primer can include the last 9 base pairs of the extracellular segment of the receptor and the first 20 base pairs of the CD3ζ segment.
         NOTE: There is no need to add a restriction site to the 5' primer since this primer will be used to generate the fused sequence, and is not used for ligation.
      2. Design a 3' primer that includes the end of the CD3ζ sequence as well as an appropriate restriction site (Figure 1C).
   3. Correct the primer sequences in each of these two reactions so that the annealing temperature is similar in each pair (note that the primer, which includes sequences of the two parts of the fusion protein, only anneals with part of the sequence in each reaction).
5. Perform an additional PCR in which a mixture of the PCR products of the first two reactions is used as the DNA template with the 5' primer of the extracellular receptor segment (Step 1.4.1.1) and the 3' of the CD3ζ segment (Step 1.4.2.2) (Figure 1D). This reaction should generate the final fused sequence.
6. Proceed as usual for cloning.
   1. Ligate the final PCR product to the target cut vector (the target vector is usually pcDNA3 which expresses G418 and ampicillin resistance).
   2. Transform the ligated vector into competent bacteria.
      1. Thaw 50 µL of the competent bacteria on ice.
      2. Add the ligated vector to the bacteria and incubate on ice for 20 min.
      3. Incubate at 42 °C for 45 s.
      4. Add 200 µL of LB without antibiotics.
      5. Incubate for 1 h at 37 °C with continuous shaking.
      6. Seed on an LB plate with the appropriate antibiotics (e.g., ampicillin solution with a final concentration of 0.1 mg/mL).
   3. Collect and grow several bacterial colonies in 5 mL of LB (in 15 mL tubes).
   4. The next day, extract plasmids with a mini-prep kit.
   5. Analyze the gene insert with restriction enzymes.
   6. Sequence the relevant colonies and verify that the sequence and the reading frame are correct (as indicated in Step 1.3).
7. Transform the verified plasmid into competent bacteria.
   1. Thaw 20 µL of the competent bacteria on ice.
   2. Add 1 µL of the plasmid to the bacteria and incubate on ice for 20 min.
   3. Incubate at 42 °C for 45 s.
   4. Add 200 µL of LB without antibiotics.
   5. Incubate for 1 h at 37 °C with continuous shaking.
   6. Seed on a bacterial growth plate with the appropriate antibiotics (e.g., ampicillin solution with a final concentration of 0.1 mg/mL).
8. The next day, grow a bacterial colony in a large LB container with 0.1 mg/mL ampicillin (~250 mL).
9. The next day, perform maxi prep according to the specific instructions provided by the kit.
   NOTE: For the electroporation procedure, a large quantity of plasmid is required, as detailed below.

2. Transfection of the Plasmid that Expresses the Chimeric Protein into BW Cells

NOTE: Different methods can also be employed for transfection (e.g., by lentiviral infection). Prior to the electroporation, perform ethanol precipitation of the DNA as described below. The next steps require sterile conditions.

1. The day before, prepare the BW5147 cells ("BW cells") for electroporation. Plate 10 plates (of 10 cm) with 10 mL of 100,000 BW5147 cells/mL (i.e., a total of 10 x 10⁶ cells) with RPMI supplemented with 10% fetal calf serum (FCS), 1% sodium pyruvate, 1% L-glutamine, 1% non-essential amino acids and 1% penicillin-streptomycin ("complete medium").
2. Place 100 µg of the pcDNA3 plasmid that expresses the fusion protein in a 1.5-2 mL tube and add 3 M sodium acetate at pH 5.3-5.5 to it. The volume of the sodium acetate should correspond to 0.1 (10%) of the volume of the 100 µg plasmid; titrate the pH of the sodium acetate with a pH meter by adding NaOH or HCl.
3. Add 2.5 volumes of 100% ethanol to the mixture of the plasmid and the sodium acetate as described in Step 2.2 (2.5x the total volume of plasmid and sodium acetate). Incubate overnight at -20 °C or for 2 h at 70 °C.
4. 24 h after the BW cells have been plated, collect all the BW cells (~100 mL) in 2 50 mL tubes. Centrifuge the cells for 5 min at 515 x g and discard the supernatant.
5. Resuspend the pellet from both tubes in 25 mL of RPMI- in a single 50 mL tube. For example, resuspend the pellet of one tube with 25 mL of RPMI- and then use this fluid to re-suspend the fluid in the other 50 mL tube so that the total pellet from the BW cells is re-suspended in 25 mL of medium in a single tube.
   NOTE: The medium should be without additions since the serum might interfere with electroporation.
6. Centrifuge the cells again for 5 min at 515 x g. Resuspend the pellet in 1 mL of RPMI, and transfer the contents to a 0.4 cm cuvette on ice.
7. Centrifuge the plasmid that underwent ethanol precipitation (Step 2.3) for 30 min at 16,000 x g and 4 °C.
8. Wash once with 1 mL of 70% ethanol and centrifuge for another 20 min at 16,000 x g and 4 °C (to wash out the salt).
9. Remove all the ethanol and let the pellet dry slightly (i.e., in an open tube in the tissue culture hood). Resuspend the pellet with 100 µL of RPMI- previously heated to 60 °C.
10. Add the re-suspended plasmid (Step 2.8) to the cells in the cuvette and incubate for 5 min on ice.
11. Electroporate the cells at 0.23 kV, 250 capacitation. This should take ~4 ms.
12. Transfer the electroporated cells to a 50 mL tube, add 50 mL of complete medium, and centrifuge for 5 min at 5,15 x g.
13. Discard the supernatant and re-suspend the cells with 50 mL of complete medium.
14. Plate the electroporated cells in 24-well culture dishes (1 mL per well, for a total of ~50 wells) and incubate at 37 °C and 5% CO₂ for 48 h.
15. Select the transfected cells with antibiotics. After 48 h add 1 mL of complete medium supplemented with 10 mg/mL G418 to each well (at this stage the final volume in each well is 2 mL and the final concentration of G418 in each well is 5 mg/mL).
    NOTE: If the plasmid contains a different antibiotic resistance, determine the antibiotic concentration required to kill the untransfected BW cells prior to this step.
16. Every 48 h, carefully discard 1 mL of the upper volume of each well without stirring the medium in the well (the BW cells tend to be at the bottom of the well). Add complete medium supplemented with 5 mg/mL G418 to each well, so that the final volume in each well is again 2 mL.
17. Examine the culture plates for cell growth in all wells regularly.
    NOTE: A change in the color of the medium to yellow may help identify growing cells; however, in the beginning, the medium will be yellow because of the G418 itself. Identify positive wells (wells with growing cells). These wells are G418 resistant and therefore are expected to express the fusion protein. This process usually takes ~3 weeks.

3. Verification of the Expression of the Transfected Receptor in Cells in the Positive Wells.

1. Stain the transfected BW cells with a specific antibody against the receptor which was transfected.
2. Compare the expression level of the receptor by flow cytometry to the expression of the control cells (untransfected BW cells).
3. After verification of one or more wells, use cells immediately for experimental purposes, grow them in culture, or freeze them for future applications.
4. Verify the expression of the transfected receptor before performing a new experiment. After a few weeks of growth, the cells with G418 with a stable receptor expression, G418 can be omitted from the culture medium.

4. Incubation of the Transfected BW Cells with Targets

NOTE: When using the transfected BW cells for the first time, it is preferable to test them on targets that express a known ligand of the receptor of interest or on plate-bound antibodies specifically directed against the receptor of interest (cross-linking experiments; see below, section 4.2.1.3).

1. Preferably, split the BW cells 24 h before the experiment (for example, by adding 10 mL of complete medium to 2 mL of cells in culture in a new 10 cm culture plate).
2. Incubate the transfected BW cells with their targets. Perform the experiment simultaneously on control BW cells that express an empty vector (or parental BW cells). 96F plates are preferable (but 96U plates can also be used). Perform the experiment in triplicate. Suspend all the cells in the complete medium.
   1. Prepare the targets. The type of target varies as a function of the objective and can be cells, cells that have been pre-incubated with antibodies, or antibodies alone. This system has also been used with bacteria as targets.
      1. If the targets are dividing cells (i.e., cell lines), irradiate them at 6,000 rad prior to the assay. Place 50,000 of the target cells in a single well of a 96 plate (in a volume of 100 µL).
      2. For experiments with antibodies and BW cells that express human FcγRs, incubate the target cells with an antibody on ice in a 96-well plate (for example, 50 µL of the target cells and 50 µL of the antibody; different antibody doses can be tested).
      3. If using antibodies alone as targets (cross-linking experiments), they should be plate-bound. For this purpose, first incubate the antibodies in a complete medium in a 96F plate for 1-2 h at 37 °C and 5% CO₂ (a typical starting dose is 0.5 μg of a specific antibody in 50 μL). Wash the plate to remove unbound antibodies.
         NOTE: In this case, a 96F plate will enable the binding of the antibody to the plate, which after the addition of the transfected BW cells will lead to the activation of the transfected receptor.
   2. Add the BW cells (effector cells). Place 50,000 BW cells in a single well of a 96 plate (in a volume of 100 µL).
   3. Complete the volume in each well to 200 µL if necessary.
   4. Incubate the plates at 37 °C and 5% CO₂ for 48 h (this time period can be calibrated).
3. After 48 h, freeze the plates at -20°C and thaw them prior to ELISA, or use them immediately for ELISA (see next steps).

5. ELISA

1. Coat an ELISA plate with an anti-mouse IL-2 antibody (anti-mIL-2). Place 0.05 µg of anti-mIL-2 in a volume of 50 µL of 1x PBS (Phosphate buffer saline) per well. Incubate the coated ELISA plate with the anti-mIL-2 antibody at 4 °C overnight or at 37 °C for 2 h.
   NOTE: To perform ELISA directly after the incubation step, the ELISA plate should be coated 24 h before the end of the incubation period of the BW cells.
2. Discard the fluid in the ELISA plate and add a blocking solution (200 µL per well) which is composed of 1x PBS and 1% bovine serum albumin (BSA). Incubate the ELISA plate for 2 h at room temperature.
3. Wash the ELISA plate three times with 0.05% PBS Tween solution (i.e., 0.5 mL of Tween-20 in 1 liter of 1x PBS).
4. Take the 96 plate which has the BW cells that were incubated with their targets (4.3). If the plate was frozen, completely thaw it (e.g., by short incubation at 37 °C). Centrifuge the 96-well plate (5 min, 515 x g) and carefully transfer 100 µL of the supernatant in each well to the pre-coated and blocked ELISA plate.
   NOTE: The supernatant should be collected from the sides of each well to avoid taking the cells.
5. If desired, add recombinant mIL-2 with defined concentrations to one of the empty rows of the coated ELISA plate to generate a standard curve of mIL-2. For example, start from an mIL-2 concentration of 2,500 pg/mL and then decrease by 50% in each subsequent well; do not add mIL-2 to the last well.
6. Incubate the ELISA plate at 4 °C overnight or at 37 °C for 2 h.
7. Wash the ELISA plate four times with PBS Tween.
8. Add biotin anti-mouse IL-2 to the ELISA plate. Use a concentration of 1 µg antibody in 1 mL of 1x PBS with 1% BSA and divide into 100 µL per well.
9. Incubate at room temperature for 1 h.
10. Wash the ELISA plate six times with PBS Tween.
11. Add HRP-conjugated streptavidin. Use a concentration of 1 µL streptavidin in 1 mL of PBSX1 with 1% BSA and divide it into 100 µL per well.
12. Incubate at room temperature for 30 min.
13. Wash the ELISA plate six times with PBS Tween.
14. Add 100 µL per well of TMB substrate solution. Complete this stage quickly to minimize differences between the wells due to time lags when adding the TMB.
15. Read the ELISA plate with an ELISA plate reader at 650 nm (the reading can be repeated if the signal is weak).