Ivis 100 imaging system

T cell-depleted (TCD) BM cells were prepared from tibia and femur of WT or MyD88-KO mice as described previously (22 (link)). In brief, splenic T cells were prepared from B6 WT mice. MHC-matched but MiHA-mismatched BALB.B mice were used as allo recipients of the 5×10⁶ TCD BM only (non-GVHD BALB.B hosts) or together with 5×10⁶ splenic T cells (GVHD BALB.B hosts). Syngeneic B6 mice (B6→B6) used as non-GVHD control. Total body irradiation was performed with split dose of 900cGy from ³⁷Cs source with 5 h interval. Acute GVHD was monitored by scoring clinical parameters as previously described (28 (link)). For BLI analysis, Luc^Tg mice backcrossed to MyD88-KO B6 or WT B6 background used as BM donors. In vivo dynamics of the engrafted TCD BM cells were longitudinally monitored using an IVIS 100 imaging system and the intensity of the emitted light was quantitated using Living image software (Perkin Elmer, Waltham, MA, USA).

Female SCID-bg mice at 4–6 weeks of age were obtained from Charles River Laboratories (Wilmington, MA, USA). Before i.v. administration, Ad5-CMV-GFP-luc (1 × 10⁸ ifu/mouse) was pre-incubated for 1 h at room temperature in the absence or presence of sCAR-CXCL12 (5 μg) in a volume of 100 μL. Three days following virus administration, mice were anesthetized with ketamine/xylazine, injected intraperitoneally with D-luciferin (300 μL; 125 mg/kg body weight), and scanned using an IVIS 100 imaging system (PerkinElmer, Waltham, MA, USA). Bioluminescence images were analyzed using Living Image software version 3.0 (PerkinElmer). Regions of interest (ROI) were drawn over the tumor and liver area, and total photon counts were calculated. Following imaging, mice were sacrificed, and livers were extracted, snap frozen, and stored at −80°C. The frozen sections were processed later for DNA extraction and determination of viral E4 copy number by real-time PCR.