Cellbrite red

To create the co-cultures, 4 day old organoids were harvested from their Matrigel droplets using dispase (1mg/ml). Organoids were washed, counted and resuspended in co-culture matrix. This co-culture matrix consisted of 1,25mg/ml Collagen-I (Corning, 354249) neutralized with neutralization buffer in a 4:1 vol/vol ratio (5X neutralization buffer = dH20|alpha MEM50mg/ml|2%NaHCO|0.1M HEPES|Ph7.5) and Matrigel (Corning). A total of 600, small organoids were plated in 20ul droplets in a 48 well plate. CAFs were trypsinized and stained with Cellbrite red (Biotium, 30023, 5ul dye/10^6 cells). After washing and counting, 28.000 stained CAFs were resuspended in 200ul co-culture medium (Table S1). For the organoid mono-culture control condition we plated 600 small organoids in co-culture matrix with ‘empty’ co-culture medium and for the CAF mono-culture condition we plated an empty co-culture matrix droplet and added 28.000 CAFs in co-culture medium. After 5 days 100ul fresh co-culture medium was added to the wells. After 8 days the co-cultures were formed and medium and/or cells were harvested for further experiments.

The LPS-stimulated RAW264.7 murine macrophage model was used in order to assess the phagocytic activity of murine RAW264.7 macrophages against SeNps-treated CT26 cells. RAW264.7 cells were seeded in glass 60 mm dishes (1 × 10⁶ cells/dish) and left to adhere overnight. Next, cells were stimulated with 1 μg/mL LPS (L4391, Simga-Aldrich) for 24 h. CT26 cells were seeded in 48-well plates at a density of 3 × 10⁴ cells/well, left to adhere overnight, and then were stained with carboxyfluorescein succinimidyl ester (CFSE, CellTrace CFSE Cell Proliferation kit, Invitrogen) according to the manufacturers’ instructions, before being treated with 15 μg/mL SeNps for 48 h. RAW264.7 macrophages (LPS-stimulated) and CT26 cells were harvested with Trypsin (Biosera) and counted. RAW264.7 cells were stained with CellBrite^® Red (Biotium, Hayward, CA, USA) according to the manufacturer’s instructions. Red-labelled RAW264.7 macrophages and green-labelled SeNps-treated CT26 cells were co-cultured at a cell ratio 2:1 in DMEM, in a humidified incubator with 5% CO₂ at 37 °C for 3 h. Cells were collected, washed in PBS and analysed with an Attune NxT flow cytometer. Data collected were analysed with FlowJo V10 software (Tree Star, Inc.). The results presented are representative of three experiments.