Clodronate liposomes (CLS), 200 μL/mouse (Liposoma BV, Amsterdam, Netherlands) were intravenously injected 48 h before LPS administration to deplete macrophages in mice23 (link),24 (link). The efficiency of depletion was confirmed using immunofluorescence analysis of the macrophages in the heart.
To overexpress Cckbr in BMMs, the cells were transfected with plasmids, 2 μg empty vector, or 2 μg pEnCMV-CCKBR (Miaolingbio), according to the manufacturer's instructions. The transfection mixture was removed 6 h post-transfection and complete medium containing 10% heat-inactivated foetal bovine serum was added. Subsequent experiments were performed 1 day after transfection. BMMs were transferred into mice, according to a published method25 (link). Briefly, 129S2/SvPas mice were intravenously injected with CLS to eliminate the macrophages. Then, after 24 h of the CLS injection, Cckbr-overexpressing BMMs (1 × 106, 200 μL) were intravenously injected into the mice. After 24 h, the mice were intraperitoneally injected with LPS (25 mg/kg, 200 μL).
To overexpress Cckbr in BMMs, the cells were transfected with plasmids, 2 μg empty vector, or 2 μg pEnCMV-CCKBR (Miaolingbio), according to the manufacturer's instructions. The transfection mixture was removed 6 h post-transfection and complete medium containing 10% heat-inactivated foetal bovine serum was added. Subsequent experiments were performed 1 day after transfection. BMMs were transferred into mice, according to a published method25 (link). Briefly, 129S2/SvPas mice were intravenously injected with CLS to eliminate the macrophages. Then, after 24 h of the CLS injection, Cckbr-overexpressing BMMs (1 × 106, 200 μL) were intravenously injected into the mice. After 24 h, the mice were intraperitoneally injected with LPS (25 mg/kg, 200 μL).