Table 1.

Cell-Based Assay Screening Protocol

Step	Parameter	Description
1	Media	50 μL DMEM with 10% serum plated in 384-well plate Corning (cat no 3712)
2	Add compounds	Use pin transfer to add 0.1 μL of compounds (5 mg/mL). Final concentration for most compounds about 10 μM
3	Plate tumor cells	96-well plate, 5×104 cells per well in 100 μL. Falcon (cat no 353072) tissue culture treated plates
4	Add compounds to 96-well plate with tumor cells	80 wells contain test compounds. 8 wells are established to generate a standard curve of known IL-2 pg/mL. 4 wells contain IFN-beta, 4 wells are untreated controls
5	Incubate	72 h in 37°C tissue culture incubator containing 5% CO2
6	Add T cells	2.5×104 cells in 50 μL per well. Spin plates gently to pellet cells and facilitate interaction
7	Incubate	24 h in 37°C tissue culture incubator containing 5% CO2
8	Take supernatant	100 μL aliquots from each well
9	IL-2 ELISA	IL-2 ELISA kit from BD Biosciences (cat no 555190). Microtest ELISA plates BD Bioscience (cat no 353279). Read OD in plate reader BioRad 3550 450–595 nm
10	Calculations	Use standard curve to calculate IL-2 pg/ml for each plate. Calculate fold increase over average of untreated control. Establish efficacy of positive control (IFN-beta) (>2-fold over control)

DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; IFN-beta, interferon-beta; IL-2, interleukin 2; OD, optical density.