Incucyte s3 analysis software

Proliferation of dermal fibroblasts was measured using the BrdU Cell Proliferation Assay Kit (BioVision). After 4 hours of BrdU incubation, cells were fixed before adding Abs and substrate. The absorbance at 450 nm was measured. In a separate experiment, the Incucyte live-cell imaging system (Sartorius) was used to monitor cell proliferation. Cells were seeded and allowed to grow overnight. After adding different treatments, cells were monitored by Incucyte up to 5 days. Cell counts were analyzed by the Incucyte S3 Analysis software (Sartorius).

CAR T cell-mediated cytotoxicity was measured through a life-cell imaging killing assay using the IncuCyte^® S3 system (Sartorius, Göttingen, Germany). For the co-cultures, 2 × 10⁴ GFP-expressing target cells (MDA-MD-231, ATCC) were seeded in a flat-bottomed 96-well plate, allowed to settle down, and incubated overnight under standard cultivation conditions (37 °C, 5% CO₂, 95% humidity) in DMEM (Biowest) supplemented with 10% FBS (Catus Biotech) and 100 µg/mL Primocin (InvivoGen). Afterwards, CAR T cells were added to the target cells at an effector:target cell (E:T) ratio of 2:1. The co-culture plate was introduced into the plate reader and four images per well were taken every 2 h for a duration of at least 4 days. Using the IncuCyte^® S3 analysis software (Sartorius, Version 2019A), the area and integrated intensity of the GFP-expressing target cells over time was analyzed.