A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

1. Preparation of Splenocyte Medium and Flow-cytometry Buffer

1. Perform all steps under a 70% ethanol-cleaned biological hood.
2. Remove 70 mL of pre-warmed Roswell Park Memorial Institute (RPMI) 1640 1x medium (commercially available and supplied in filtered 500 ml volume sterile bottles) and place in a sterile tube. The removed medium will be used later in the protocol.
3. Supplement the remaining 430 mL of RPMI 1640 1x with 50 ml inactivated fetal bovine serum (FBS), 5 mL penicillin/streptomycin, 5 mL N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 5 ml non-essential amino acids, 5 mL sodium pyruvate, and 0.05 mL filtered (1M) 2-mercaptoethanol.
   NOTE: Pre-warm splenocyte medium using a water bath set at 37 ºC to avoid heat shocking cells. This is important to avoid induction of cellular apoptosis.
4. To prepare the flow-cytometry buffer, add 2 mL of FBS to 98 ml phosphate buffered saline (PBS) and keep refrigerated until use (preferably within three days).

2. Generation of Splenocyte Cell Suspension from Nur77^GFP Mouse Spleens

1. Aseptically isolate the spleen from a 6-8 week old female Nur77^GFP mouse.
   1. To achieve this, work under a sterile hood. Soak the sacrificed mouse fur, scissors, and forceps in 70% ethanol.
   2. Lay the mouse on its right side, and cut the skin and muscles in the upper left abdominal quadrant. Inspect the incision area to visibly locate the spleen then cut it out. Keep the spleen in splenocyte medium on ice until ready to perform the next step.
2. Place the spleen(s) in a 10 cm² cell culture petri dish containing 5 ml of pre-warmed splenocyte medium.
3. Mash the spleen using a sterile syringe plunger until the solution is turbid. A spleen collagen matrix should remain by the end of the procedure.
4. Following extensive mashing in splenocyte medium, collect the cell suspension and pass it through a 70 µm cell strainer placed on a 50 mL tube to filter out any debris or clots.
5. Centrifuge at 500 x g for 5 min. After discarding the supernatant, re-suspend the cell pellet in 2-3 mL of in-house produced or commercial red blood cell lysis buffer. Following pipetting (2-3 times) let the suspension rest for 20-30 s.
6. Add 5 mL of PBS or a suitable buffer of choice then centrifuge the cell suspension for 5 min at 500 x g.
7. Remove the supernatant (should be red in color as it contains lysed red blood cells) and re-suspend the cell pellet in 2 ml of splenocyte medium.
8. Using a hemocytometer, count the number of cells stained with trypan blue to differentiate between live and dead (necrotic) cells.

3. T-cell Isolation from the Splenocyte Cell Suspension

1. Centrifuge the cell suspension for 5 min at 500 x g. Resuspend the cells in serum-free RPMI medium (50 ml from step 1.3) to obtain a cellular concentration of 10 x 10⁶ cells/mL.
2. Transfer the cells to a 5 mL polystyrene tube and set aside a 100 µL aliquot of splenocytes for purity assessment/comparison at the end of the purification step.
3. Add normal rat serum to the cell suspension at 50 µL/mL followed by a T cell isolation antibody cocktail (50 µL/mL).
4. Mix the cell suspension and let it rest for 10 min. This step allows the antibody cocktail to bind all unwanted cells.
5. Add the streptavidin rapid spheres (magnetic beads) for 2.5 min, then bring the volume to 2.5 mL using the serum-free medium.
6. Place the tube in a cell isolation magnet for 3 min.
7. Transfer the T cell suspension into a new polystyrene tube by holding the magnet and pouring out the solution in one move.
   NOTE: The isolated suspension contains the purified T cells. All unwanted cells are held on the side of the tube bound to the magnetic streptavidin beads.
8. Count the isolated T cells using trypan blue and a hemocytometer.
   NOTE: At this step, the purity of the isolated T cells can be verified (optional) by analyzing the percentage of CD3⁺ events before and after T cell isolation by flow cytometry.
   1. Briefly, centrifuge 1 ml of splenocyte cell suspension at 500 x g. Discard the supernatant and suspend the cells in a flow cytometry buffer at a concentration of 1 x 10⁶ cells/mL.
   2. Add fluorescent-tagged anti-mouse CD3 antibody at a concentration of 1:100. Incubate at 4 °C for 30 min. Wash once, centrifuge, and re-suspend in flow cytometry buffer (400 µL) for analysis by flow cytometry.

4. T-cell Activation and the Induction of green fluorescent protein (GFP) Expression

1. Seed the T cells, in suspension, into a round-bottomed 96-well plate at a concentration of 2.5 x 10⁵ cells/well.
2. Add recombinant interleukin (IL)-7 at 2 ng/mL and CD3/CD28 magnetic beads (25 µL/10⁶ cells). Keep a portion of the T cells untreated with beads to serve as a negative control for later measurements that represent non-activated T cells.
3. Twelve hours later, harvest the T cells from the 96-well plate by gently pipetting the cell suspension in each well up and down to break any bead-cell complex/aggregates. Collect the suspension from all wells into a 5 mL polystyrene tube.
4. Place the tube containing the suspension inside the same cell isolation magnet used previously for the purification of T cells and allow it to rest for 5 min.
5. Transfer the T cell suspension into a new polystyrene tube by holding the magnet and pouring out the solution in one move.
6. Centrifuge the cells at 500 x g for 5 min. Re-suspend the cells in fresh splenocyte medium to get a concentration of 2 x 10⁶ cells/mL.
7. Assess GFP expression intensity by flow-cytometry (optional for quality control) at 12 to 24 h post-stimulation in comparison with non-activated T cells.¹²
   NOTE: The GFP fluorescence is intrinsic to the Nur77^GFP T cells and is manifested upon successful activation of the T-cell receptor, TCR.
8. For assessment of viability and activation (optional) by microscopy, stain the activated cells with Hoechst 30 min prior to analysis at a concentration of 0.2 µg/ml. Add an adequate volume of the cell suspension to a cover slide or a well of a flat-bottomed black-sided 384-well plate and examine under a fluorescence microscope to assess living cells. Dead cells will not retain nuclear staining.

5. High Throughput Screening (HTS) of Small Molecules

1. Prepare a cell suspension at 2 x 10⁶ cells/mL of the activated T cells (magnetic beads or antibodies/CD40L) or non-activated groups.
2. Plate 75,000 cells/well in a 384-well plate (volume of 40 µL). Use a flat-bottomed black-sided 384-well plate or a microscope cover slide for viability and activation assessment by fluorescent microscopy (optional quality control step prior to HTS) using Hoechst stain (refer to steps 4.8 for details).
3. Manually or using an automated system, add the drugs of choice (dissolved in 0.5% dimethyl sulfoxide, DMSO) to each well.
4. Add the vehicle (DMSO) to positive (activated) and negative (non-activated) control wells. Adjust DMSO concentration to a maximum of 0.5%.
5. Incubate the plates for 24 h (or incubation time of choice) at 37 ºC and 5% carbon dioxide, CO₂.
6. On the day of the screening, dilute the Hoechst 33342 stain solution (1:3,333; for e.g. add 10 µL to the cells in the 384-well plates to generate a total volume of 50 µL). Stain 30 min before GFP analysis by adding Hoechst solution to achieve a concentration of 0.2 µg/mL.
7. Gently pipette the cells up and down to obtain a homogenous distribution in each well.
   NOTE: This step is important in the case of dispensing the drugs (step 5.3) using an automated system as the cells tend to accumulate at one side of the well, opposite to the direction of the flow.
8. Spin the plates at 45 x g for 3 min at room temperature.
9. Leave the plates to rest for 15 min at room temperature.
10. Perform plate(s) read-outs, using the automated confocal high content screening (HCS) system. Load the plate to the machine. Set the objective at 40X or higher magnification. Use camera #4 for Hoechst (UV lamp) and camera #1 for GFP (laser 488). Set up the machine to read 6-10 fields per well. Adjust the machine to do two sequential readings per field at 488 nm (to read GFP) and UV light (to read Hoechst). Set the objective at 40X or higher magnification.
    NOTE: The system is a computerized microscope that does not require any adjustments. The focal distance, the intensity of the incident light, and the time of exposure are all set up automatically by the machine.