24 well multi dish culture plate

RAW 264.7 cells of a macrophage cell line were seeded on each well of 24 well multi-dish culture plate (Corning Inc., Corning, NY) at a density of 1 × 10⁴ cells/cm² and cultured in 500 μl of Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen Corporation, Ltd., Carlsbad, CA) with 10 vol% fetal calf serum (FCS) for 24 h. Next, the medium was exchanged to DMEM, and then the DAR-4M micelles and free DAR-4M in DMEM were added to each well. After 3 h incubation, the number of cells proliferated was evaluated with a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium (WST-8) assay kit (Nacalai Tesque. Inc., Kyoto, Japan).

RAW264.7 cells were seeded on glass bottom dish (Matsunami Glass Ind.,Ltd., Osaka, Japan) at a density of 1 × 10⁵ cells/cm² and cultured in 1 ml of DMEM medium with 10 vol% FCS for 24 h. The medium was exchanged to DMEM containing 100 ng/ml of LPS, and incubated further for 24 h. Next, the medium was exchanged to FCS-free DMEM, and then, free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin, or DAR-4M micelles were added to each dish. After 1 h incubation, cells were washed with PBS. The cells were viewed on a Nikon ECLIPSE 90i confocal laser scanning microscope (Nikon Corp., Tokyo, Japan).
To evaluate the fluorescent intensity of cells incubated for 1 h with free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin, and DAR-4M micelles, RAW264.7 cells were seeded on each well of 24 well multi-dish culture plate (Corning Inc., Corning, NY) at a density of 1 × 10⁵ cells/cm² cultured in 1 ml of DMEM with 10 vol% FCS for 24 h. Then, the medium was exchanged to FCS-free DMEM, and free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin or DAR-4M micelles were added to each well. After 1 h incubation, cells were washed with PBS. The cells were viewed on a Nikon ECLIPSE 90i confocal laser scanning microscope (Nikon Corp., Tokyo, Japan).