Two days before transduction, bone marrow cells were harvested from 8 to 12 week old OT-I transgenic mice and cultured at 1.5X107 cells per 10 cm plate in 15 ml DMEM supplemented with 15% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin, 10mM hepes, 20 ng/ml murine interleukin-3 (IL-3), 50 ng/ml human IL-6 and 50 ng/ml murine stem cell factor (SCF) (R&D Systems). The concentrated virus was transduced into the pre-cultured BMCs. After 48 hours incubation bone marrow cells were collected and washed. Sub lethally irradiated (800 rads) WT B6 recipients were injected IV with 4 × 106 bone marrow cells in PBS. Splenocytes from these BM chimeras were harvested 6–8 weeks post-transplant and were enriched by negative selection using a CD8a+ T cell Isolation Kit II (Miltenyi Biotec,). Purity of CD8a+ T cells was over 80%. Cells were then stained with anti-CD8 Pac Orange, anti-Thy1.1 PerCP, and anti-ICOS APC and CD8+ Thy1.1+ ICOS+ cells were purified by FACS sorting on a BD FACS Aria. Post-sort ICOS-OT-I T cell populations were over 95% pure.
Murine stem cell factor
Manufactured by R&D Systems
Murine stem cell factor (SCF) is a cytokine that promotes the survival, proliferation, and differentiation of hematopoietic stem and progenitor cells. It is a key regulator of hematopoiesis and is essential for the maintenance and expansion of various blood cell lineages.
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2 protocols using murine stem cell factor
1
Generation and Isolation of ICOS+ OT-I T Cells
2
Single Cell Culture Protocol for Murine Hematopoietic Stem Cells
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For single cell cultures, single murine CD34- CD48- CD150+ Lineage- SCA1+ KIT+ (LSK) cells (CD34- SLAM cells) were sorted directly into a 96-well round-bottomed plate on an Astrios high speed cell sorter (Beckman Coulter). Single cells were deposited with an efficiency of 90%. These CD34- SLAM cells comprise both CD34- and CD34low populations, as these have been reported to contain similar frequencies of long-term repopulating cells.12 (link) These plates were preloaded with 100 mL of filtered (0.20 mm, Sartorius) CM supplemented with murine stem cell factor (SCF: 100 ng/mL) and murine interleukin-11 (IL-11; 20 ng/mL) (two growth factors; 2GF), both from R&D Systems (BioTechne). Additional cultures were further supplemented with Col1 (300 μg/mL; BioVendor) and human NGF (250 ng/mL; R&D Systems- BioTechne) (four growth factors; 4GF). Every 24 h, the number of cells per well was enumerated using a light microscope. After 5 days, cells that had divided at least once were harvested, pooled, stained with antibodies, and analyzed by flow cytometry. In some experiments, colony-forming ability of pools from divided cells was assessed using standard assays (M3434; Stemcell Technologies).
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