Xenogen ivis 200 in vivo imaging system

To study the ability of CD8⁺ iTregs in preventing GVHD, recipient mice were lethally irradiated at 700 cGy for BALB/c or 1,100 cGy for BDF1 (split dose). Irradiated mice were adoptively transferred with CD8⁺ iTregs 1 × 10⁶ /mouse for BALB/c or 2 × 10⁶ /mouse for BDF1 together with 5×10⁶ TCD-BM from C57BL/6 (WT). Three days later, CD25-depleted T-cells (Teffs) from WT were injected to induced GVHD; 0.7×10⁶ for BALB/c or 3×10⁶ for BDF1. Recipient survival rate, GVHD clinical score, and body weight were followed for 80 days. To study in GVL model, MLL-AF9-GFP leukemic cells 2×10⁴ /mouse or P815 mastocytoma 2.5–5 ×10³/mouse were infused on the day of BMT. MLL-AF9 is a mixed lineage leukemia including myeloid leukemia expressing CD11b; therefore, MLL tumor growth was monitored in peripheral blood of recipient mice and determined for CD11b⁺GFP⁺ cells by flow cytometry (34 (link), 35 ). P815 growth was measured with bioluminescent imaging (BLI) using Xenogen-IVIS®200 in vivo Imaging System (Perkin-Elmer). Pacritinib (PAC) was administered at 100 mg/kg/day/mouse by oral gavage for 4 weeks. The mice were monitored for survival rate and tumor mortality.

Recipient mice were lethally irradiated at 700 cGy for BALB/c and 1000-1200 cGy (2 split doses, 3-hour interval) for B6 or BDF1 recipient mice using an X-RAD 320 irradiator (Precision X-Ray). Lethally irradiated BALB/c, B6 or BDF1 mice were transplanted with BM from B6 or FVB donors with or without T cells at doses indicated. Recipient survival was monitored throughout the experiment. The development of GVHD was monitored twice per week for weight loss and once per week for clinical signs of posture, skin damage, hair loss, ruffled fur, diarrhea, and decreased activity (Nguyen et al., 2018a )( Cooke etal., 1996). To evaluate the GVL response, tumor cells were injected i.v. on the same day of transplantation. In case of luciferase transduced P815 (5000 cells/mouse), tumor burden was estimated with bioluminescent imaging (BLI) using Xenogen-IVIS 200 in vivo Imaging System (Perkin-Elmer, Waltham, MA). MLL-AF9-GFP⁺ tumors were identified by measuring via the percentages of GFP⁺ cells in peripheral blood using flow cytometry. Recipient survival and GVHD severity demonstrated by recipient weight loss were monitored throughout experiment.

As previously describled (5 (link)), recipient mice were lethally irradiated at 700 cGy for BALB/c and 1000-1200 cGy (2 split doses, 3-hour interval) for B6 recipient mice using an X-RAD 320 irradiator (Precision X-Ray). Within 24 hours of irradiation, Balb/c or B6 recipient mice were transplanted with 5.0×10⁶ T-cell depleted bone marrow cells (TCD-BM) from B6 or FVB donors with or without T cells (0.5-1×10⁶/mouse). Recipient survival was monitored throughout the experiment. Body weight loss was monitored twice per week and clinical signs of GVHD were monitored once per week and include posture, skin damage, hair loss, ruffled fur, diarrhea, and decreased activity(5 (link)) (24 (link)). For GVL experiments, tumor cells were injected intravenously (i.v.) on the same day of transplantation. In cases of luciferase transduced acute myeloid leukemia C1498 (2000 cells/mouse), tumor burden was estimated with bioluminescent imaging (BLI) using Xenogen-IVIS 200 in vivo Imaging System (Perkin-Elmer, Waltham, MA). MLL-AF9-GFP⁺ tumors were identified via the percentages of GFP⁺ cells in peripheral blood using flow cytometry. For human-to-mouse xenograft models, sublethally irradiated NSG-A2⁽⁺⁾ mice were transplanted with HLA-A2⁽⁻⁾ PBMCs (15×10⁶). Recipient survival and GVHD severity demonstrated by recipient weight loss were monitored throughout experiment.