Rmccl2

MSCs were seeded and cultured with or without recombinant murine CCL2 (10 ng/mL, rmCCL2, R&D systems) (Figure 2A); (1) MSCs, without any intervention; (2) Tem-rmCCL2+MSCs, unaltered control MSCs incubated with temporal rmCCL2 stimulation for the initial 1 day of assay; (3) Con-rmCCL2+MSCs, unaltered control MSCs incubated with continuous rmCCL2 stimulation for the whole period of assay; (4) virus⁺MSCs, MSCs infected with empty lentivirus vector; (5) CCL2⁺MSCs, MSCs infected with murine CCL2 secreting lentivirus vector. These groups of MSCs were used for the following experiments. In addition, direct co-culture of MSCs with naïve primitive macrophages (1:1 ratio mix of each type of cells and basal medium) was also performed to investigate the effect of CCL2 on the cell-cell interaction between MSCs and macrophages.

To prepare BMDMs, BM cells were isolated from 6- to 8-week-old male C57BL6 mice and VDUP1-KO mice and maintained as previously described manner [70 (link)]. For qRT-PCR, BMDMs were incubated with 1 µg/mL LPS for 8 h. For the chemotaxis assay, 10 ng/mL rmCCL2 (R&D Systems, Minneapolis, MI, USA) was placed in the bottom chamber of a 24-well plate. BMDMs were seeded at a density of 1 $\times$ 10⁵ cells/well in the upper chamber (5 µm, Corning Incorporated, NY, USA). Each assay condition was performed in triplicate. After 18 h, the migrated cells were fixed with 4% paraformaldehyde, and the membranes were stained with 0.5% crystal violet (w/v) dissolved in 20% methanol (v/v) in PBS for 20 min. Five random views were photographed in each well at 100 $\times$ magnification with a microscope. The number of migrated cells in five random fields was counted, and data from each membrane were averaged.

Spleen T lymphocytes (3 × 10⁶ in HBSS without Ca²⁺/ Mg²⁺) were placed in the upper chamber of 5.0 μm pore diameter transwell tissue culture inserts (BD Falcon, USA). Transwell inserts were placed in the individual wells of a 24-well cell culture plate containing assay buffer or lung homogenates from naïve, sham-operated and CLP-operated mice, neutralized (30 min, 37 °C) with anti-CCL2 mAb (2.5 ng/well), anti-CCL3 mAb (200 ng/well) or anti-CCL5 mAb (50 ng/well). The recombinant chemokines rmCCL2 (2.5 ng/well), rmCCL3 (4 ng/well) and rmCCL5 (4 ng/well) (R&D Systems, USA) were used as positive controls. After 2 h, the migrated cells were counted, labeled as described above, and analyzed by FACScalibur. Results are expressed as chemotactic index, generated by using the number of cells that migrated towards buffer as comparison.