Coomassie brilliant blue g 250 reagent

PSL labeled with the fluorescent dye was used to examine the uptake of PSL by macrophages. The amount of fluorescent dye was 1 mol％ of total lipid. Raw 264.7 cells were maintained in Dulbecco s modified Eagle medium (Wako, Osaka, Japan) and incubated at 37℃ in a humidified atmosphere containing 5％ CO 2 . Media were supplemented with 10％ heat-inactivated fetal bovine serum (FBS) , penicillin (100 U/mL) , streptomycin (100 μg/ mL) and amphotericin B (0.25 μg/mL) ( all from Gibco, Invitrogen Co., Grand Island, NY, USA) . Raw 264.7 cells (5× 10 4 ) were grown in 24-well plates at 37℃ for 24 h and were further incubated at 37℃ for 1 or 24 h after adding PSL. After 1 and 24 h, the cells were washed three times with PBS and lysed in 200 μl of lysis buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2.5 mM MgCl 2 and 0.05％ NP-40 substitute) . Fluorescence intensity was detected using a microplate reader (Synergy HT, BIO-TEK Instruments Inc., Winooski, VT, USA) . Total protein concentration was determined by the Bradford method (Coomassie Brilliant Blue G-250 reagent; BIO-RAD Lab., Hercules, CA, USA) and detected by absorbance at 595 nm. The results are presented as the fluorescence intensity per mg of total protein.