25 cm2 culture flasks

Human umbilical cord blood-derived MSCs were generously provided by Xin’An Stem Cell Engineering Co., LTD (Hefei, Anhui, China). Informed consent for the donation of the MSCs used in this study was obtained prior to parturition. The isolated MSCs were cultured in DMEM (Hyclone, USA), supplemented with 10% (v/v) FBS (Hyclone, USA). The cells were incubated in a humidified atmosphere at 37 °C with 5% CO₂, for which the MSCs were seeded on 25 cm² culture flasks (Eppendorf, Hamburg, Germany). The medium was changed every other day till the cells reached 80–90% confluence. Later, 1×PBS (Hyclone, USA) was added to remove dead cells, followed by adding trypsin (Gibco, Carlsbad, CA, USA) to detach the cells. Detached cells were then centrifuged at 100 g for 5 min for immediate use or seeded into new culture flasks for passaging and use in future experiments. Early passages (passages 1–6) of MSCs are used in the study.

COLO320 cells (American Type Culture Collection; ATCC, VA, USA) were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA) medium supplemented with an antibiotic-antimycotic cocktail (100 U/mL penicillin and 100 μg/mL streptomycin; Life Technologies, Carlsbad, CA) and 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) under a humidified atmosphere of 5% CO₂ in air at 37°C. Cells ( 0.5–1.0×10⁶) were seeded into 25 cm² culture flasks (Eppendorf AG, Hamburg, Germany) and passaged at 70–80% confluency. To assess the effect of puromycin on cell viability, 0.02–0.1 × 10⁵ cells were seeded in RPMI containing 10% FBS (100 μL/well) in a 96-well cell culture plate (Eppendorf AG, Hamburg, Germany). All cultures were performed in triplicate. After 12 h, the medium was replaced with RPMI 1640 medium with 10% FBS that contained 0–20 μg/mL puromycin (Life Technologies, Carlsbad, CA). Cells were then allowed to incubate for 48 h at 37°C before cell viability was determined using the CellTiter 96^® AQ_ueous One Solution Reagent assay (Promega, Madison, WI) per the manufacturer’s protocol.
Cells were passaged using 0.25% trypsin-EDTA followed by counting (ViaStain AOPI Staining Solution,cridine orange/propidium iodide Nexcelom Bioscience, Lawrence, MA) using an automated cellometer (Cellometer K2, Nexcelom Bioscience, Lawrence, MA) and then harvested at 90% confluency.