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

Begin with a fluorescent-compatible multi-well plate containing a suspension of transgenic mouse-derived T lymphocytes.

These lymphocytes contain a green fluorescent protein or GFP expression cassette, controlled by T cell receptor or TCR engagement with immunomodulatory compounds.

Treat each well with a solution containing immunomodulatory compounds with different potentials.

During incubation, immunomodulatory compounds interact with TCRs on T lymphocytes.

TCR interaction with a stimulating compound triggers the activation of the gene cassette, elevating GFP expression.

In contrast, TCR interaction with an inhibitory compound inhibits the gene cassette and reduces the GFP expression.

Next, overlay the cells with nucleic acid fluorescent dye to stain the nuclei.

Centrifuge the plate to settle the cells for accurate fluorescence measurement.

Under a fluorescence microscope, viable T lymphocytes exhibit two fluorescence signals corresponding to cell nuclei and green fluorescent proteins.

The intensified green fluorescence signifies successful stimulation of T lymphocytes, while a faint intracellular green fluorescence signal confirms the inhibitory effect of immunomodulatory compounds on T lymphocytes.

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 4 for the UV lamp and then set camera number 1 for laser 488. Load the plate to the high-content screening system. Next, set the automated confocal high-content screening system to read 6 to 10 fields per well, with two sequential readings per field at 488 nanometers and UV light.