and the remaining leukemic cells were preserved in liquid nitrogen until use. Leukemic repopulated cells were thawed and washed, resuspended in RPMI containing 10% fetal bovine serum, 5mM MgCl2 and 0.2 mg/ml DNase I and incubated at 37 1C for 10 min. Cells were washed OSU-03012 742112-33-0 and resuspended at 1 million cells per ml in RPMI containing 20% fetal bovine serum with cytokines, and incubated with imatinib for 48 h at 37 1C in a CO2 incubator. In an in vitro long term culture, spleen cells derived from leukemic NOG mice were co cultured with S17 stromal cells and treated with imatinib and everolimus.16 S 17 cells and leukemic cells were passaged twice weekly. Reagents Everolimus and imatinib were supplied by Novartis Institutes for Biomedical Research. Imatinib was dissolved in dH2O and used for in vitro and in vivo experiments.
Received 10 December 2010, revised 27 March 2011, accepted 4 April 2011 Correspondence: XAV-939 284028-89-3 Dr Y Minami, Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsurumai cho, Showa ku, Nagoya 466 8550, Japan. u.ac.jp Citation: 1, e17, doi:10.1038/bcj.2011.16 & 2011 Macmillan Publishers Limited All rights reserved 2044 5385/11 www.nature.com/bcj Everolimus was stored as 10 2M stock solution in dimethylsulfoxide for an in vitro experiment. For in vivo experiments, everolimus was formulated at 2% in a microemulsion vehicle. Aliquots of everolimus and control vehicle were stored at 20 1C. Immunoblotting Antibodies against the phospho Abl, p CrkL, p mTOR, p p70 S6 kinase, p 4EBP1, MCL 1, p AKT, AKT and p FOXO1/FoxO3a were from Cell Signaling.
Immunoblotting was performed with the standard protocols as previously described.17 Flow cytometric analysis and cell sorting After the treatment period, cells were washed at 4 1C and then stained with anti CD34 allophycocyanin, anti CD38 PECy7, and anti CD45 APC Alexa Fluor 750 antibodies for 30 min on ice. Cells were subsequently labeled with annexin V fluorescein isothiocyanate and propidium iodide according to the manufacturer,s protocol. The cells were acquired by FACS Aria and analyzed by Flow Jo software. DNA contents analysis was assessed using the standard procedure as previously described.18 For CD34t selection, leukemic cells were subjected to immunomagnetic separation using a MACS CD34 MicroBead Kit following the manufacturer,s recommendations.
The collected cells were applied to a second column and the purification step was repeated. Staining of cells with Hoechst 33342 with PyroninY was performed as previously described.19 Briefly, thawed leukemic spleen cells were separated with the MACS CD34 MicroBead Kit into CD34t cells and flow through cells containing CD34 cells. MACS separated cells or drug treated cells on S17 were washed and stained with Hoechst 33342 and PyroninY, and washed at 4 1C. MACS separated CD34t cells were then stained with anti CD38 APC, and flow through cells containing CD34 cells were stained with anti CD34 APC. Flow cytometric analysis was performed using FACS Aria. For cell sorting, leukemic spleen cells were stained with anti CD34 APC, anti CD38 PE Cy7 and anti CD45 APC Cy7 antibodies and labeled with PI. PI CD45t cells were sorted for CD34 and CD38 expression using FACS Aria, incubated with treatment drugs for 6 h at 37 1C in a CO2 incubator as described above. Mouse models Humanized leukemic mouse