Computational protocol: Single-Cell STAT5 Signal Transduction Profiling in Normal and Leukemic Stem and Progenitor Cell Populations Reveals Highly Distinct Cytokine Responses

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Protocol publication

[…] CB CD34+ cells were grown in HPGM (Cambrex, Walkersville, MD) supplemented with 100 ng/ml Stem Cell Factor (SCF; Amgen, USA), FLT3 Ligand (FL; Amgen, USA) and Thrombopoietin (TPO; Kirin, Tokyo, Japan) at 5×105 cells/ml at 37°C and 5% CO2. After 3 days, cells were washed twice with PBS and cytokine-depleted in HPGM at 8×105 cells/ml overnight at 37°C. Primary AML MNCs and PB CD34+ cells were suspended in HPGM at 1.5×106 cells/ml for 2 hours at 37°C. Cells were stimulated with cytokines for 15 minutes at 37°C after which fixation was performed with 2% PFA at a 1∶1 dilution directly into the medium at room temperature for 10 minutes. Cells were washed with 2% BSA/PBS twice and permeabilized with 1 ml 90% methanol on ice for 30 minutes. The permeabilized cells were washed twice, followed by FcR blocking (1 µl per 5×105 cells) at 4°C for 10 minutes. Samples were stained in 100 µl final reaction volumes with the following antibodies at room temperature for 1 h: phospho-STAT5-Alexa 647; CD34-FITC; CD38-Percp/Cy5.5; CD123-PE; CD45RA-PE-Cy7. Samples were washed twice after incubation and analyzed on an LSR II flow cytometer (BD). Data were analyzed using WinList 6.0 (Topsham, ME, USA) and FlowJo (TreeStar, OR, USA) software.The K562 cell line was cultured in RPMI 1640 medium (Biowhittaker, Verviers, Belgium) supplemented with L-glutamate, 10% fetal calf serum (FCS; Sigma) and 1% penicillin/streptomycin (P/S). The UT-7 cell line was cultured in IMD medium (PAA Laboratories GmbH, Pasching, Austria) with 10% FCS and 1% P/S, supplemented with 10 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF; Genetics Institute, Cambrigde). The UT-7 cells were stimulated with 10 U/ml Erythropoietin (EPO; Cilag; Belgium) as indicated in the text. Cells were used for STAT5 phosphorylation by FACS as described above. […]

Pipeline specifications

Software tools WinList, FlowJo
Application Flow cytometry
Diseases Leukemia