Transwell inserts with polycarbonate membrane with 3-μm pore size (Corning) were coated with fibronectin (10 μg/ml) in HBSS overnight at 4°C, followed by blocking with 1% BSA in HBSS for 1 hour at RT. The conditioned media were collected from gel-encapsulated human MSCs that are cultured in the presence or absence of TNFα (100 ng/ml) for 1 day, followed by washout and cultured in the absence of TNFα for another day. The same cell number per gel (~50,000 per 8-mm disc) was used to collect the conditioned media. Fresh human peripheral mononuclear cells (~100,000) were added to the top wells in serum-free FluoroBrite DMEM, while the conditioned media were added to the bottom wells. In some experiments, the conditioned media were incubated with the neutralizing antibody against human CCL2 (MAB679, R&D Systems) or the mouse IgG2B isotype control (MAB004, R&D Systems) for 3 hours at 4°C prior to the migration assay. The cells were allowed to migrate for 3 hours at 37°C, 5% CO2. The cells collected from the bottom wells were subject to flow cytometry analysis for different lineages using the following anti-human antibodies: FITC CD14 (HCD14), APC CD4 (RPA-T4), APC-cy7 CD8 (SK1), PE CD19 (HIB19), and PE-cy7 CD56 (HCD56), all purchased from BioLegend. The cell number per sample was counted by adding a predefined number of polystyrene beads (Spherotech). The fraction of migrated cells was calculated by dividing with the total number of cells for each lineage added to each well.

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