Nuclightred

SHP-77, 22Rv1 or C42B cancer cells well labeled with NucLightGreen or NucLightRed (Sartorius) as indicated. On day 0, 3–5 ×10³ cells were plated and left overnight to attach. On day 1, cells were treated with NT control BiTE^® (5 nM), AMG 757 (5 nM), or AMG 160 (400 pM), and co-cultured with activated T cells, which were plated at an effector-to-target ratio of 5:1 or 10:1. The plates were loaded into an IncuCyte S3 Live Cell Analysis System (Sartorius), and images were obtained every 4–6 hours over 5 days.

For IC₅₀ assays and combination index (CI) measurements, transduced cells with NucLight Red (4476, NucLight Lentivirus, Sartorius) were plated at 10,000 cells/well in a 96-well plate. Cells were treated with 1 μmol/L of ADI-PEG20 (Polaris), increasing doses of DTX (S1148, Selleckchem), and increasing doses of GEM (S1714, Selleckchem), either alone or in combination, in phenol red-free MEM (11966-025, Thermo Fisher). Cell death was determined using an IncuCyte FLR imaging system (Sartorius). For cell death assays, cells were plated in medium containing 50 nmol/L YOYO-1 (Thermo Fisher Y3601). Cells were treated as described, imaged every hour over a period of 3 days, and analyzed using IncuCyte image analysis software (Sartorius). For quantification of death, YOYO-1 florescence was normalized to Nuclear Red counts for each respective well. For quantification of proliferation assays, only Nuclear Red counts were used. Once IC₅₀ values were established, cell death assays were performed as previously stated, except cells were treated with 1 μmol/L of ADI-PEG20, 3.32 μmol/L DTX, and 5.42 μmol/L GEM alone or in combination. All inhibition studies used 100 μmol/L of a c-Myc inhibitor, 10058-F4 (F3680, Sigma-Aldrich) 1 hour prior to treatment and maintained within culture medium throughout the experiment.

1x10⁴ RPMI-8226 myeloma cells transduced with NucLight Red (Sartorius) were seeded into 96-well ultra-low binding plates (Corning). Cells were cultured for 48-72 hours to allow for spheroid formation. Next, 4x10⁴ iNK cells were gently added to each well with or without daratumumab at a final concentration of 10 μg/ml, and cells were co-cultured for 5 days. At the end of the culture, cells in each well were disrupted into a single cell suspension and stained with a fluorescently conjugated CD56 antibody and fixable viability dye for flow cytometry analysis. Tumor cells were quantified based on NucLight Red, and iNK cells were quantified based on CD56 expression.