4.10. In Vitro Bystander Killing Assay

SKBR-3 and A549 cells were seeded in single culture or co-culture in 96-well plates at a density of 8000 cells per well and incubated overnight at 37 °C under a 5% CO₂ atmosphere. The incubation media was removed and 100 µL of a 10 nM solution of tested conjugate in DMEM complete medium were added to the plate and incubated for 5 days. Cells were then collected and transferred into a round-bottom 96-well plate suitable for high-throughput flow cytometry. Cells were rinsed 3 times with PBS, resuspended in 50 µL DPBS, and incubated for 30 min at room temperature in the dark with anti-HER2 APC-conjugated antibody (BD Bioscience, Cat#340554) and eBioscience Fixable Viability Dye eFluor 780 (Thermo Scientific, Waltham, MA, USA, Cat#650865-14). Flow cytometry analysis was performed using a BD Fortessa flow cytometer controlled by BD FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (BD Bioscience).