A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

The overall goal of this florescence-based lymphocyte assay is to identify new small molecule immunomodulators. This method can help answer key questions in the field of immunology about the mechanism of lymphocyte activation and inhibition. The main advantage of this phenotypic function and assay, that it can be adapted for drug discovery.

Demonstrating the procedure will be Ahmed Fouda, a graduate student from our laboratory. Under a sterile work hood, disinfect the fur of a six to eight week old female Nurr77 GFP mouse with 70%ethanol, and place the animal on its right side. Next, make an incision in the skin and muscles of the upper left abdominal quadrant, and locate and remove the spleen, placing the tissue into splenocyte medium on ice.

When all of the spleens have been collected, transfer the tissues to a 10 centimeter squared cell culture dish containing five milliliters of prewarmed splenocyte medium, and use a sterile syringe plunger to mash the tissues until the solution is turbid. Following extensive mashing in splenocyte medium, filter the cell suspension through a 70 micron cell strainer into a 50 milliliter tube to remove any debris, and centrifuge the resulting single cell suspension. Resuspend the pellet in two to three milliliters of red blood cell lysis buffer, sopping the reaction with five milliliters of RPMI, or another suitable buffer, after 20 to 30 seconds.

Then collect the cells with another centrifugation, and resuspend the pellet in twp milliliters of splenocyte medium for cell counting. To isolate the T cells, centrifuge the cells again, and resuspend the pellet in serum free RPMI medium at a one times 10 to the seven cells per milliliter concentration. Transfer the cells to a five milliliter polystyrene tube, and set aside a 100 microliter aliquot of splenocytes to be used later for purity assessment.

Next, add 50 microliters per milliliter of normal rat serum to the cells, followed by the addition of 50 microliters per milliliter of T cell isolation antibody cocktail with gentle mixing. After 10 minutes, incubate the cells with streptavidin conjugated magnetic beads for two and a half minutes. Then, add serum free medium to the cells for a final volume of two and a half milliliters, and place the tube in a cell isolation magnet for three minutes.

At the end of the incubation, holding the tube in the magnet, pour the supernatant into a new polystyrene tube in one move. The T cells can then be counted. Seed T cells into a round bottom 96-well plate at a concentration of 2.5 times 10 to the fifth cells per well containing 200 microliters of fresh splenocyte medium per well.

Add recombinant interleukin 7 and CD3/CD28 magnetic beads to the appropriate wells, and place the plate in a cell culture incubator. 12 hours later, gently triturate the cell suspension in each well to break up any bead cell aggregates. Then transfer the cell suspensions from each well into individual five milliliter polystyrene tubes.

When all of the cells have been collected, place each tube of cells into the separation magnet for five minutes at a time, and decant the supernatants when the tube still inside the magnet, into new polystyrene tubes at the end up each incubation. Then collect the cells by centrifugation, and resuspend the pellets in fresh splenocyte medium at two times 10 to the sixth cells per milliliter concentrations. For small molecule high-throughput screening, plate 40 microliters of cells per well in a 384-well plate, and treat the appropriate wells with the drug or vehicle of choice for the desired treatment period in the cell culture incubator.

30 minutes before the GFP analysis, stain the cells with the appropriate concentration of Hoechst solution, thoroughly distributing the stain with gentle pipetting. When all of the wells have been dyed, centrifuge the plates to collect the cells at the bottom of the wells, and allow the cells to rest for 15 minutes at room temperature. Load the plate according to the manufacturer’s instructions.

Then, set the objective at 40X or higher. Set camera number four for the UV lamp, and then set camera number one for laser 488. Load the plate to the high content screening system.

Next, set the automated confocal high content screening system to read six to 10 fields per well with two sequential readings per field, at 488 nanometers and UV light. CD3 positive T cells represent approximately 30%of a given mouse spleen, demonstrating a 10%basal GFP expression. Following CD3/CD28 T cell receptor bead stimulation, a five to six fold increase in the percentage of GFP expression is observed.

Likewise, CD19 positive B cells represent 50 to 55%of the total splenocytes with the basal GFP expression similar to that of non activated T cells. Interestingly, B cell receptor stimulation using anti-mouse IgG, IgM, and recombinant CD40L, triggers only a trivial increase in GFP expression. After magnetic bead isolation as just demonstrated, the isolated CD3 positive T cells still display a 10%basal GFP expression.

CD3/CD28 bead stimulation however, induces a significant upregulation of the GFP response. Similarly, after B cell magnetic bead isolation, the purified activated B cells exhibit a higher expression of GFP compared to activated B cells in splenocytes. In this representative experiment, primary high-throughput screening of 4398 compounds revealed several activators and inhibitors of T cell activation.

But the observed GFP expression of the stimulated T cells, 20 times higher than that measured for the unstimulated T cells. Allowing the generation of a window of action zone where the GFP inhibitory compounds could easily be detected. Once mastered, this technique can be completed in 36 to 48 hours if it is performed properly.

While attempting this procedure, it is important to remember to count the cells after each step. Following this procedure, other methods like ELISA or western blotting can be performed to answer any other additional questions. This technique will pave the way for drug discovery immunomodulation in rodents.

After watching this video, you should have a good understanding of how to isolate and activate T or B cells derived from the Nurr77 GFP mouse plates.