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