Cell culture inserts for 6 well plates

MSCs were seeded at 4.8 × 10 4 cells/well in 6-well plates (Falcon, Corning, NY, USA), and RAW264 cells were seeded at a density of 2.4 × 10 4 cells/well in Cell Culture Inserts for 6-well plates (0.4 μm pore size, 1.0 × 10 6 pores/cm 2 ) (Falcon) and co-cultured without cell contact. When the MSCs reached confluence, the cell culture inserts were discarded, MSCs were collected, and total RNA was extracted and purified using β-mercaptoethanol (Sigma-Aldrich Japan, Tokyo, Japan). A Simpli Amp Thermal Cycler (Applied Biosystems, Foster City, CA, USA) was used to obtain cDNA from total RNA. Using the prepared cDNA as a template, gene expression of CCL2 was determined by using the SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) and Light Cycler 480 II (Roche Diagnostics, Basel, Switzerland). The primer sequences are listed in Table 1. The reaction conditions employed were 45 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 10 s. The gene expression of CCL2 in MSCs cultured alone was evaluated by quantitative real-time PCR as a control. Gene expression values were normalized to control values. The fold increase in gene expression by MSCs cultured alone was monitored, and the mRNA level was normalized to that of GAPDH.

RAW264 cells were seeded at a density of 2.4 × 10 4 cells/ well in 6-well plates, and MSCs were seeded at 4.8 × 10 4 cells/well in Cell Culture Inserts for 6-well plates (0.4 μm pore size, 1.0 × 10 6 pores/cm 2 ) (Falcon) and co-cultured without cell contact. When RAW264 cells reached confluence, the Cell Culture Inserts were discarded and the RAW264 cells were collected; CCR2 mRNA expression was analyzed using quantitative real-time PCR as described above. The gene expression of CCR2 in RAW264 cells cultured alone was evaluated as a control. The primer sequences employed are listed in Table 1. The fold increase in gene expression by RAW264 cells cultured alone was monitored, and the mRNA level was normalized to that of GAPDH. Forward: ATT CTC CAC ACC CTG TTT CG Reverse: GAT TCC TGG AAG GTG GTC AA using Cell Culture Inserts for 24-well plates (8.0 μm pore size, 1.0 × 10 5 pores/cm 2 ) (Falcon) migration assay.