8 μm pore inserts

Cells were seeded in trans-well chambers with 8-μm-pore inserts (Corning, NY, USA) for migration and invasion assays. For the migration assay, cells in serum-free medium were plated in uncoated inserts and incubated for 24 h. For the invasion assay, the inserts were coated with 100 μL of Matrigel (BD Biosciences, NJ, USA), and cells were plated in the serum-free medium described above for an incubation period of 24 hours. 500 µL culture medium containing 15% FBS was added to the lower chamber. The cells attached to the bottom of the membrane were fixed with 4% paraformaldehyde, then subjected to being stained with 5% crystal violet (Sigma-Aldrich), and counted at 200× magnification. The assays were repeated for three times.

Transwell migration assays were performed in 24 mm transwell plates with 8 μm pore inserts (Corning, USA). Cell were put into the upper chamber of the transwell inserts with no-serum medium, and complete medium with 10% serum and C-C motif chemokine 2 (CCL2) at a concentration of 20 ng/ml was added to the lower chamber. After the cells migrated for 24 h, cells on the inner of the membrane were wiped off and on the outer of the membrane were fixed by 4% PFA for 15 min and stained by crystal violet (Beyotime Biotechnology, Shanghai) for 30 min. The number of migrated cells was quantified by counting the average number of cells from 5 random fields at the microscope.

For the migration assay, 4 × 10⁴ cells (200 μL serum-free cell suspension) were seeded into the upper Transwell chamber with 8 μm pore inserts (Corning, NY, USA), while the bottom chamber was filled with 600 μL RPMI 1640 medium supplemented with 10% fetal bovine serum. After incubation at 37°C with 5% CO₂ for 24 h, cells invading the lower surfaces were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet stain solution, while cells on the upper surface were scraped. Nine random fields were used for statistical analysis. For the invasion assay, 1 × 10⁵ cells (200 μL serum-free cell suspension) were seeded into the upper Transwell chamber, which was prepaved with Matrigel. The remaining steps were the same as those for the migration assay.

To compare the migration ability of cells, hCPCs were seeded at a density of 2 × 10⁵ cells on a 12-well plate (Thermo Scientific) and they were grown until they attained 100% confluence. Using a sterile yellow pipette tip, a scratch was created in each well. After 8 h of incubation, the wound distance of the cells was visualized at ×40 magnification using a light microscope (Olympus). For transwell migration assays, DMSO (young: using less than passage 8, senescent: using greater than passage 17) and long-lasting treated hCPCs (passage 17) were suspended in serum-free F12 Ham’s medium and placed in 8 μm pore inserts (Corning) to migrate toward complete hCPC culture medium. After 24 h of incubation, the cells were removed from the medium and fixed at room temperature for 10 min with 4% paraformaldehyde. The membrane was stained at room temperature for 30 min with 0.005% crystal violet in 20% MeOH solution. Upper surface unmigrated cells were removed by a cotton swab. The cell migration ability was quantified by enumerating the cell count in 10 random 200 × microscopic fields.

Transwell migration and invasion assays were performed using 8-μm-pore inserts (Corning Incorporated Costar, Tewksbury, USA). For the migration assay, cells (5 × 104 cells per well) were seeded into the upper chamber directly with serum-free medium. For the invasion assay, the inserts were coated with 50 µL of Matrigel (Solarbio, Beijing, China), and 600 µL culture medium containing 15% FBS was added to both lower chambers. Migrated or invaded cells were stained with 0.1% crystal violet 24 h after culture and counted in three random fields under light microscopy in a 100 × scope.

Cells were suspended in a medium without FBS and seeded in 8 μm pore inserts (Corning, USA) at a density of 2 × 10⁴ cells per insert. The inserts were placed into 24-well plates containing 800 μL of 30% FBS medium. After culturing for 48 h, the cells on the upper side of the filter were fixed with 4% formaldehyde for 30 min and stained with 2% crystal violet for another 30 min. The cells on the upper side were then wiped with swabs, while the cells on the lower side were imaged randomly under a microscope.