3D Cell Culture Viability Assay

The plates used were 96-well plates with a U-shaped bottom and a less adherent surface. Cells were seeded onto a plate at a concentration of 3000 cells/well. The plates were centrifuged at 2000 rpm for 5 min, and then the prepared cells were left for 3 days in an incubator (37 °C, 5% CO₂) to form a 3D structure [9 (link)]. On the fourth day, treatment was performed. After 48 h, 5 μL of calcein and 1 μL of propidium iodide (IP) from a Cellstain double staining kit (Sigma Aldrich, Darmstadt, Germany) were added to each well. Fluorescence intensity was measured using the Agilent BioTek Lionheart FX Automated Microscope (Agilent Technologies, Santa Clara, CA, USA) at excitation (about 501 nm) and emission (about 521 nm) for calcein and at excitation (about 488 nm) and emission (about 617 nm) for IP.