Ap20187

AP20187 (B/B Homodimerizer, Takara Bio USA) was dissolved in 100% ethanol to form a stock solution at a concentration of 62.5 mg/ml and stored at −20 °C. A dosing solution of 10 mg/kg active drug was prepared from stock solution, PEG-400 (100%), and Tween (2%) per manufacturer instructions. A solution (vehicle control) containing of ethanol, PEG-400 (100%), and Tween (2%) was prepared similarly. AP20187 or vehicle control were dosed at 2 μl solution/g mouse per leg (5 μg/g AP20187). The dosing solutions were used within 30 min of preparation.
Liposomes were stored at 4 °C. Prior to injection, the clodronate-containing liposomes (5 mg/ml) and PBS-containing liposomes (vehicle control) were removed from the refrigerator and allowed to acclimate to room temperature (18 °C). The tubes containing liposomes were inverted 8–10 times to ensure even distribution. Liposomes were dosed at 20 μl of solution per leg (100 μg clodronate). In the ALI mouse model, mice were injected 1 day before and 1 day following lung injury.
The mouse fur was removed from bilateral lower limbs overlying the tibialis anterior (TA) muscle compartment. Skin was cleaned with 70% ethanol and the dosing solution injected in four aliquots along the body of the TA muscle compartment, as previously described (Files et al., 2012 (link)).

Macrophages were ablated in male C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J mice, also known as macrophage Fas Ligand-induced apoptosis (MaFIA) mice (Jackson Strain #005070). MaFIA mice (10–12 weeks old) were treated with 5mg/kg AP20187 (Takara Bio USA) in 1% DMSO + 10% PEG-400 + 2% Tween-80 in sterile water daily for 3 days, then every other day through the end of the study to induce dimerization of Fas and subsequent apoptosis in colony stimulating factor 1 receptor promoter (Csf1r)-expressing cells [49 (link)]. Untreated age-and sex-matched MaFIA mice were used as controls.