Fig. 2.

ClodL selectively reduces macrophage viability in vitro and in vivo. (a) Graded dose–response treatments with Clodronate (0 to $0.1\mathrm{mg}/\mathrm{mL}$) or PBS control liposomes demonstrated that at all Clodronate concentrations tested, only macrophage RAW264.7 cells, but not nonmacrophage E0771 breast tumor cells or HFF-1 fibroblasts, were sensitive to Clodronate depletion ($n=8$ each data point, $p<0.05$ for RAW264.7 PBSL versus ClodL at all doses). For each cell type, fluorescent intensities were standardized to the PBS control liposome group (no ClodL), to account for differences in proliferative rate inherent to cell type. Liposomes containing PBS (vehicle) only also did not affect proliferation of RAW264.7 macrophages at any liposome concentration (RAW264.7/PBSL trace). (b) Flow cytometric analysis indicated that endogenous $\mathrm{F}4/{80}^{+}$ TAMs were depleted by in vivo Clodronate treatment in both wild-type (wt) and TNF-${\alpha }^{(-/-)}$ mice [$n=21\mathrm{wt}/\mathrm{PBSL}$; $n=16\mathrm{wt}/\mathrm{ClodL}$; $n=15$ TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$; $n=15$ TNF-${\alpha }^{(-/-)}/\mathrm{ClodL}$]. Moreover, these data also indicate that under baseline (PBSL, i.e., no depletion) conditions, $\mathrm{F}4/{80}^{+}$ macrophage populations were not significantly different in wt versus TNF-${\alpha }^{(-/-)}$) animals.