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

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.