96 well microplates

Optimal cell densities for a miniaturized clonogenic assay in 96-well microplates (Starlab, Hamburg, Germany) were determined as described [29 (link)]. The following seeding numbers per well were chosen: 80 cells for HuCCT1 and OCUG-1, 50 cells for KKU-055, 40 cells for EGI-1, and 30 cells for NOZ and the MMNK-1 cells. The seeding of OZ and TFK-1 cells at different cell numbers did not result in clonogenic growth. Therefore, these cell lines were excluded from the experiments. The determination of optimal cell density was carried out in biological and technical triplicates.
For the investigation of clonogenic growth, cells were seeded according to the determined optimal seeding numbers in 96-well microplates and were grown overnight. Then, cells were washed with DMEM without serum and incubated with different concentrations of tazemetostat in DMEM with serum using a 1:2 dilution series (starting concentration 80 µM, 10 steps) for seven days. To avoid evaporation, empty spaces on the plate were filled with DPBS. Confluence was measured after seven days with the Spark multimode reader.

Recombinant FasL (2 μg) (PeproTech, 310-03H) was immobilized onto 96-well microplates (Starlab, CC7672-7596) overnight at 4°C and the plate was washed several times with PBS. For the proliferation experiments, 5 × 10⁴ CAR T cells was seeded onto the FasL-immobilized microplate and incubated for 5 days. The number of CAR T cells was quantified by flow cytometry, using CountBright Counting Beads. For the measurement of NF-κB activity, 1 × 10⁵ transduced NF-κB Jurkat reporter cells were seeded onto the FasL-immobilized microplate, incubated overnight, and then treated as described.