Chambers with 8 μm pores

Macrophage migration was evaluated with a transwell cell migration assay as previously described [15 (link)]. Briefly, the chambers with 8 μm pores (Corning Costar, USA) were firstly pretreated with DMEM/F12 medium. Then, 100 μL of DMEM/F12 containing 1 × 10⁵ macrophages and 1% FBS was added into the upper chamber, and the lower compartment was filled with 600 μL medium containing 10% FBS without cells. After incubation for 18 h, the transwell membrane was fixed with 4% paraformaldehyde (PFA) (Macklin, China) for 30 min. After removing the nonmigrated cells from the upper chamber with a cotton swab, the migrated cells were stained using a 0.1% crystal violet solution for 30 min. For quantification, 6 pictures of each membrane were photographed with an inverted microscope (Leica, Germany).

To determine the tropism of AF-MSCs and IFNα-AF-MSCs for HeLa cells in vitro, the Transwell migration assay was performed as previously described [13 (link)]. HeLa cells were cultured for 24 h in 1640 medium containing 10% FBS; after being trypsinized, HeLa cells were resuspended and plated at 1 × 10⁶/2 ml in the lower wells of Transwell chambers and allowed to adhere for 24 h. After being cultured in α-MEM (0.5% FBS) for 24 h, AF-MSCs and IFNα-AF-MSCs were plated in the upper well of chambers with 8 μm pores (Corning Costar, USA) at a density of 1 × 10⁵/800 μl. HSF, cultured in DMEM + 10% FBS previously, were used as control. These chambers were incubated at 37°C for 24 h to allow the MSCs to migrate. Then, the cells that had not migrated were removed from the upper chamber with a wet cotton swab, and the cells that had migrated were fixed and stained with trypan blue. Images of the stained cells that had migrated to the bottom of the Transwell chamber through the upper membrane were obtained using a Leica DMI4000B inverted phase-contrast microscope (Leica, Germany). The number of cells in 4 high-power fields (×200) per membrane was counted manually.