Isolation and Transduction of Murine HSCs

Bone marrow cells were isolated and cultured as previously described^{28 (link)}. In short, femurs, tibias, hips, and spines were isolated from donor mice, crushed, and lysed with Ammonium-Chloride-Potassium (ACK) lysing buffer (Gibco Cat# A1049201). LSK cells (lineage^– Sca-1⁺ Kit⁺) were enriched with a CD117 MACS isolation kit (Miltenyi Cat# 130-091-224) and then sorted to purity. The LSK cells were spin transduced with lentiviral vectors on Retronectin-coated plates (Takara Bio Cat# T100B) at a multiplicity of infection (MOI) of 25. LSK cells were then transferred intravenously into irradiated CD45.2⁺ wild-type recipients.

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LaFleur M.W., Nguyen T.H., Coxe M.A., Miller B.C., Yates K.B., Gillis J.E., Sen D.R., Gaudiano E.F., Abosy R.A., Freeman G.J., Haining W.N, & Sharpe A.H. (2019). PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nature immunology, 20(10), 1335-1347.

Publication 2019

ACK) lysing buffer CD117 MACS isolation kit Retronectin-coated plates

Ammonium Ammonium chloride Bone marrow cells Buffer Cells Chloride potassium Donor Femurs Hips Infection Isolation Mice Potassium Retronectin Sca 1 Spines Tibias Vectors

Corresponding Organization :

Other organizations : Boston VA Research Institute, Harvard University, Dana-Farber Cancer Institute, Brigham and Women's Hospital

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Variable analysis

independent variables

Lentiviral vector transduction of LSK cells
Multiplicity of infection (MOI) of 25

dependent variables

Measurement of outcomes after transfer of transduced LSK cells into irradiated CD45.2+ wild-type recipients

control variables

Femurs, tibias, hips, and spines isolated from donor mice
Crushing and lysis of bone marrow cells with Ammonium-Chloride-Potassium (ACK) lysing buffer
Enrichment of LSK cells (lineage- Sca-1+ Kit+) using CD117 MACS isolation kit
Sorting of LSK cells to purity
Spin transduction of LSK cells on Retronectin-coated plates

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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