Nunc culture plates

An evaluation of the cytotoxicity induced by CPP-iCAL36 conjugates was performed using a Cytotoxicity Detection KitPlus (LDH, (Merck, Fontenay Sous Bois, France) following the manufacturer’s instructions. In brief, 1.6 × 10⁴ cells (160 μL) were seeded in 96-well Nunc culture plates (Thermo Fisher Scientific Inc., Rockford, IL, USA). Then, 48 h post-seeding, cells were rinsed twice with D-PBS and then incubated with 160 μL of a 1 μM, 10 μM, or 100 μM CPP-iCAL36 solution (in triplicates) in OptiMEM (Life Technologies) for 24 h at 37 °C with 5% CO₂. At least 3 wells were kept for the LDH negative control (0% viability) and for non-treated cells as a positive control (100% viability).
After a 24 h incubation period, negative controls were performed by adding Triton X-100 (Sigma-Aldrich, Saint-Quentin-Fallavier, France) (~10 min incubation at 37 °C). Afterward, 50 μL of supernatant was collected, mixed with 50 µL of the “dye solution/catalyst” mixture, and incubated in the darkness for 30 min at room temperature. The reaction was stopped by adding 25 µL of HCl (1 N) to each well before measuring the absorption at 490 nm. Relative toxicity (%) = ((exp. value − value non-treated cells)/(value triton − value non-treated cells)) × 100.

For studying the multicellular resistance of tumor cells to TRAIL-induced apoptosis in vitro, spheroids were used. Formation of multicellular spheroids was performed in 96-well Nunc culture plates (Thermo Scientific, Waltham, MA, USA) coated with 1.5% agarose [25 (link)]. After plating 5 × 10³ cells in 200 µL of growth medium per well the cultures were incubated for 96 h at 37 °C in a humidified atmosphere of 5% CO₂.

24 well Nunc culture plates (Thermo Fisher Scientific) were coated with 10 μg/mL of anti-LAIR-1 agonist (clone Dx26) (6 (link)) or 10 μg/mL of mouse isotype control IgG1 (eBioscience-Thermo Fisher Scientific) diluted in PBS overnight at 4°C. A total of 1 × 10⁶ PBMCs or 0.5 × 10⁶ purified monocytes were seeded in the pre-coated plates with complete medium after incubation with Fc receptor blocking reagent (Miltenyi Biotech). Cells were pre-incubated for 2 h at 37°C in a 5% CO₂ incubator and then either left unstimulated or stimulated with LPS-TLR4 ligand (100 ng/mL, Invivogen) or IFN-α2a (1,000 U/mL, Cell Sciences). PBMCs were stimulated overnight at 37°C in a 5% CO₂ incubator and then harvested for flow cytometry staining. Monocytes were stimulated for 5 h at 37°C in a 5% CO₂ incubator and afterwards supernatants were collected and stored at −80°C and cells were lysed with RLT buffer (Qiagen) and stored at −20°C until further analysis.