Interleukin 3 (il 3)

5-FU primed bone marrow cells were incubated in X-Vivo 15 (Lonza, Allendale, NJ) supplemented with 50 ng/ml SCF, 50 ng/ml TPO, 10 ng/ml IL-3 and 10 ng/ml IL-6 (all from Peprotech, Rocky Hill, NJ) for 24 hours. After incubation, cells were spin-infected with retroviral supernatant supplemented.

C-kit⁺ BM stem and progenitor cells were isolated from donor mice and transduced with retroviruses as previously described (Somervaille and Cleary, 2006 (link)). For triple transductions, BM cells were spinoculated sequentially with each retroviral supernatant for 30 minutes at 32°C and 1350g. Transduced cells (3-10×10⁵) were transplanted by retro-orbital injection immediately following spinoculation into lethally irradiated (900 cGy) syngeneic recipient mice, together with an additional radioprotective dose of 2×10⁵ nucleated whole BM cells. An aliquot of the post-spinoculation cells was cultured in methylcellulose medium with 20 ng/ml SCF, 10 ng/ml IL-6, 10 ng/ml GM-CSF and 10 ng/ml IL-3 (Peprotech, Rocky Hill, NJ), in the presence or absence of G418 or puromycin drug selection, to determine retrospectively the transplanted dose of transduced CFCs.
For congenic transplantation analyses, post-spinoculation CD45.2⁺ cells were serially replated in semi-solid culture for three rounds, as previously described (Somervaille and Cleary, 2006 (link)). 8×10⁵ pooled, washed cells were then transplanted into sub-lethally irradiated CD45.1⁺ recipients (450 cGy).
For mouse leukemia CFC assays and limit dilution secondary transplantation assays, splenocytes frozen in 10% DMSO were thawed and then rested in RPMI with 20% fetal calf serum and 20% WEHI-conditioned medium for four hours. FACS sorted Mac1⁺ PI^- cells were then plated in semi-solid culture, as above, for six days prior to colony enumeration, or injected in varying doses into sub-lethally irradiated syngeneic recipients. For secondary transplantation of cells derived from a single AML CFC, individual AML colonies were plucked from semi-solid culture after five days, replated once and then, after a further five days, 2.5×10⁵ cells were washed and injected into sub-lethally irradiated syngeneic recipients. For secondary transplantation of AML cells transduced with Myb knockdown or control lentiviruses, cells were sorted by FACS 48 hours following transduction for red fluorescence and then transplanted into sub-lethally irradiated syngeneic mice.

Mouse S1pr2 or the control truncated Ngfr were inserted in a retroviral vector containing a cytoplasmic-domain truncated human CD4 reporter^{50 (link)}. An insert containing a HA tag and the 14-amino acid myristoylation site of human v-src at the N-terminus of Akt1 was inserted into the MSCV2.2 retroviral vector containing GFP downstream of the IRES. This construct was also inserted into a Thy1.1 reporter vector containing a loxP-eGFP-stop-loxP cassette upstream of the insertion site, and BM from Cr2-cre mice was transduced. MD4 B cells were transduced as previously^{8 (link)}. For S1pr2, due to effects on cell viability or homing, ~3-fold more cells were activated and transduced. For BM transductions, donor mice were injected i.p. with 3 mg 5-Fluorouracil (Sigma). BM was harvested after 4 days and cultured in DMEM containing 15% FBS, antibiotics (50 IU/ml penicillin, 50 µg/ml streptomycin; Cellgro) and 10 mM HEPES (Cellgro), supplemented with IL-3, IL-6, and stem cell factor (SCF) (each at 0.1 µg ml⁻¹, Peprotech). Cells were spin-infected twice at day 1 and 2 and transferred to irradiated recipients on day 3.