Living image acquisition and analysis software version 4

We created PDX models that developed stable bioluminescence by infecting T-ALL and B-ALL cells with a lentivirus expressing both GFP and luciferase. Then, we transplanted these cells into mice that were used later to perform bioluminescence imaging. The lentivirus was produced in HEK293 cells after transduction with Lipofectamin 2000 (Thermo Fisher Scientific) of the pCCLc-MNDU3-Luciferase-PGK-EGFP-WPRE vector (Addgene, #89608), as well as PAX2 (Addgene, #12260) and pCMV-VSV-G (Addgene, #8454) plasmids. After two days, viral supernatants were recovered, and six-well plates were incubated 4 h with retronectin (Takara, Ozyme). Viral supernatants were then spinoculated for 30 min at 4,000 g. Cells were cultured on these plates for three days in StemMACS media (Miltenyi Biotech). Lentiviral transduced cells (GFP⁺) were sorted on a FACSAriaIII cell sorter (BD Biosciences) and transplanted in NSG mice to generate bioluminescent PDX models. Animals were injected with potassium salt of D-luciferin (150 mg/kg body weight). Following isoflurane-induced anesthesia, animals were imaged 20 min after D-luciferin injection using an IVIS Lumina III system coupled to Living Image acquisition and analysis software version 4.0 (Perkin Elmer).

We created PDX models that developed stable bioluminescence by infecting B-ALL cells #2 with a lentivirus expressing both GFP and luciferase [67 (link)]. Then, we transplanted these cells into mice that were used later for bioluminescence imaging. The lentivirus was produced in HEK293 cells after transduction with Lipofectamin 2000 (Thermo Fisher Scientific) of the pCCLc-MNDU3-Luciferase-PGK-EGFP-WPRE vector (Addgene, #89608), as well as PAX2 (Addgene, #12260) and pCMV-VSV-G (Addgene, #8454) plasmids. After 2 days, viral supernatants were recovered, and 6-well plates were incubated for 4 h with retronectin (Takara, Ozyme). Viral supernatants were then spinoculated for 30 min at 4000×g. Cells were cultured on these plates for three days in StemMACS media (Miltenyi Biotech). Lentiviral transduced cells (GFP⁺) were sorted on a FACSAriaIII cell sorter (BD Biosciences) and transplanted in NSG mice to generate bioluminescent PDX. Following isoflurane-induced anesthesia, animals were imaged 15 min after d-luciferin (Merck) injection, at 150 mg/kg body weight, using an IVIS Lumina III system coupled with Living Image acquisition and analysis software version 4.0 (Perkin Elmer).