Assessing Liposomal Clodronate Cytotoxicity

A murine macrophage cell line, RAW264.7, was purchased from American Type Culture Collection (Manassas, VA) and was cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). Liposomal clodronate (lipo-CL2MDP) was kindly gifted by Dr N. van Rooijen [11 (link), 12 (link)]. Liposomal PBS (lipo-PBS) was used as a control. RAW264.7 cells treated with the indicated concentrations of lipo-CL2MDP or lipo-PBS were seeded at a density of 10,000 cells/well in a 96-well plate and incubated for 48 hours at 37°C. After the indicated time, the viability of the cells was assessed by a colorimetric assay (TetraColor One®; Seikagaku Co., Tokyo, Japan) as described elsewhere [10 (link), 13 (link), 14 (link)]. Briefly, 10 μL of TetraColor One® was added to each well, and the mixture was incubated for an additional 4 hours to measure the viability of cells. Absorbance at 450 nm was monitored and the IC₅₀ values of the cells were calculated.