Blocking agent

A previously trypsinized suspension of 10⁶ cells was resuspended in 125 µl of PBS containing 2% FBS and 5 mM EDTA. This suspension was then blocked by adding 12.5 µl of blocking agent (Miltenyi Biotec) and incubated for 10 min on ice. An anti-CD133 antibody conjugated to the fluorochrome phycoerythrin (PE) (MACS) was then added at a 1:25 dilution and incubated for 30 min on ice in the dark. A tube containing 1–106 cells was left unlabeled with the antibody to use as a negative control for staining. After the end of the incubation period, the cells were washed 2 times with PBS containing 2% FBS and 5 mM EDTA. Subsequently, the cells were centrifuged for 5 min at a speed of 1000 rpm (112 g) and resuspended in 500 µl of PBS containing 2% FBS and 5 mM EDTA. Finally, the cell suspension was examined on a Canto II analytical cytometer (BD Biosciences), with the cells separated into positive and negative populations based on staining for the CD133 marker in certain cases.

The cells were maintained in culture with or without treatment for 48–72 h (Table S1). Then, both suspended and adherent cells were collected. 1 × 10⁶ of those cells were resuspended in PBS with 2% FBS and 5 mM EDTA. Next, the cells were incubated with blocking agent (Miltenyi Biotec) for 10 min at 4 °C, and then, antibody labeling (Miltenyi Biotec) was performed for 15 min at room temperature and 15 min at 4 °C. Following this, two washes were performed using PBS with 2% FBS and 5 mM EDTA. After that, the previously described kit (Inmunostep) was used to label dead cells. Finally, the cells were examined using a FACSCanto II flow cytometer (BD Biosciences), and the results were analyzed using Diva software. The number of dead cells was measured in EpCAM + cells and the whole population. All cells stained with propidium iodide were considered dead.

RAW264.7 cells and BMDMs were collected with a scraper, blocked with a blocking agent (Miltenyi Biotech, Germany) for 10 min, and then incubated with a PE-conjugated anti-mouse CD86 (1:200) or FITC-conjugated anti-mouse F4/80 (1:200) antibody based on the manufacturers’ instructions. F4/80 was utilized to identify macrophages, and CD86 was utilized as the marker of M1 macrophages. The expression of CD86 was calculated from the fluorescence intensity. All data were collected by flow cytometry (ACEA NovoCyte, China) using Novo Express (ACEA NovoCyte, China) and calculated using FlowJo software version X (Tree Star, USA).