Mab679

Quantification of chemokines in cell supernatants was done by ELISA. The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and biotinylated polyclonal goat anti-human CXCL8 (BAF208) antibodies purchased from R&D Systems (Minneapolis, MN, USA) [26 (link)]. Similarly, the human CCL2 ELISA was also developed in our laboratory with reagents from R&D Systems [monoclonal mouse anti-human CCL2 (MAB679) and biotinylated monoclonal mouse anti-human CCL2 (BAF279)] [28 (link)]. Human CCL3 and murine CCL2, CXCL1 and CXCL6 were measured with a specific Duoset ELISA kit following the manufacturer's instructions (R&D Systems).

Cell migration was quantitated in duplicates in a 24-well Transwell using polycarbonate filters with 5 μm pores (Corning Costar 3421). Monocytes (0.3 × 10⁶ cells in 200 μl of 80% DMEM and 20% RPMI 1640, both containing 10% FBS and 1% Penicillin/Streptomycin) were added to the upper compartment of the insert. The lower well contained 600 μl of the test media (as described in Fig. 6). After a 2-h incubation at 37°C, the inserts were gently removed, the cell-containing media were gently homogenized, and 400 μl were used to count cells by flow cytometry (Cytoflex). All samples were collected during a fixed time of acquisition and gated for live singlets. The data were analyzed by Kaluza Analysis Software (Beckman Coulter).
The experiment was repeated with PBMCs in the top well (as described in Fig. S5). Monocytes could be distinguished from lymphocytes according to the physical parameters of the flow cytometry scatter (separation of the two populations based on forward scatter (FSC) and side scatter (SSC), after gating for live singlets).
In some experiments, the media were pre-incubated with the neutralizing antibody against human CCL2 (R&D Systems MAB679, 2 μg/ml) for 1.5 h at room temperature prior to being used in the lower chambers of the migration assays.

For CCL2 detection, A3250-control or A3250-shCCL2 tumor cells (~7 × 10⁶ cells) were cultured in 5 ml serum-free media for 24 hr. Conditioned media were concentrated 23-fold using Amicon Ultra Centrifugal Filter Units (Ultracel-3K, Millipore) and 20 μl was loaded on a 12% SDS-PAGE gel. Western blot analysis^{52 (link)} was performed^{52 (link)} using anti-human CCL2 (R&D Systems MAB679) at 1 μg/ml. Secondary antibody, Alexa Fluor 680 goat anti-mouse IgG (Invitrogen, A-21059) was used at 1:10,000 dilution and signal was detected using the LI-COR Odyssey fluorescence imaging system. Western blot source data is provided as a Source Data file.

Monocyte migration assays were performed using polycarbonate membrane Transwell inserts (5.0-μm pores, 24-well plate, Corning). A total of 3 × 10⁴ MSCs in 600 μL of DMEM or cell-free culture supernatant were seeded in the lower chambers. After adhesion, MSCs were treated for RNA interference or lentiviral transfection. Then, the culture supernatant was replaced with FBS-free culture supernatant with or without 0.5 μg/mL anti-MCP1 neutralizing antibody (MAB679, R&D Systems), and 1 × 10⁶ monocytes in 100 μL of FBS-culture supernatant were seeded in the upper chambers after staining with CFSE for 15 minutes. After 12 hours, the culture supernatant in the lower chambers was collected, and the number of monocytes in the supernatant was calculated by flow cytometry. CFSE-positive cells were regarded as migrated monocytes in the lower chambers. The molecular biological studies of MSCs were also performed after coculture with monocytes.