Myeloid Precursor Expansion and Differentiation Assay

Myeloid hematopoietic precursors were expanded from BM cells using granulocyte-monocyte-colony stimulating factor (GM-CSF), stem cell factor (SCF) and leukaemia inhibitory factor (LIF) for 2 to 3 days, following published conditions [8 (link), 30 (link)]. Then, myeloid precursors were seeded on two L8 cell culture chambers for the xCELLingence RTCA monitoring system (ACEA biosciences), at a density of 200000 cells per well. DC or B16-MDSC differentiation medium was added to myeloid precursors, and treatments were carried out simultaneously in duplicates. After 30 min, the indicated chemical inhibitors were added at concentrations reported to be cytotoxic to cancer cells. Control wells were treated with carrier solution (either water or DMSO). The following inhibitors were used: AKT inhibitor X (Calbiochem), tyrosine kinase inhibitor TX-1123 (Calbiochem), MEK inhibitor PD0325901 (SIGMA), ERK inhibitors SCH772984 and VTX-11e [31 (link)], broad PKC inhibitor GÖ 6983 (Santa Cruz Biotechnology), PKC inhibitor NPC-15437 dihydrochloride (Santa Cruz Biotechnology), selective LCK and FYN inhibitor PP2 (Santa Cruz Biotechnology), and the SRC and FYN inhibitor Saracatinib (MedChem Express). IC50s for each inhibitor were calculated using the RTCA data and analysis software, using duplicates for each drug treatment.

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Gato-Cañas M., de Morentin X.M., Blanco-Luquin I., Fernandez-Irigoyen J., Zudaire I., Liechtenstein T., Arasanz H., Lozano T., Casares N., Chaikuad A., Knapp S., Guerrero-Setas D., Escors D., Kochan G, & Santamaría E. (2015). A core of kinase-regulated interactomes defines the neoplastic MDSC lineage. Oncotarget, 6(29), 27160-27175.

Publication 2015

MEK inhibitor PD0325901 Saracatinib

Cancer Cell culture Cells Dmso Drug Granulocyte colony stimulating factor Hematopoietic Inhibitors Leukaemia inhibitory factor Mdsc Mek inhibitor pd0325901 Monocyte Npc 15437 Rtca Saracatinib Sch772984 Stem cell factor Tx 1123

Corresponding Organization :

Other organizations : University College London, Navarrabiomed, Universidad de Navarra, Genomics England, University of Oxford, Goethe University Frankfurt

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Protocol cited in 3 other protocols

Variable analysis

independent variables

Differentiation medium (DC or B16-MDSC)
Chemical inhibitors (AKT inhibitor X, tyrosine kinase inhibitor TX-1123, MEK inhibitor PD0325901, ERK inhibitors SCH772984 and VTX-11e, broad PKC inhibitor GÖ 6983, PKC inhibitor NPC-15437 dihydrochloride, selective LCK and FYN inhibitor PP2, and the SRC and FYN inhibitor Saracatinib)

dependent variables

IC50 values for each chemical inhibitor

control variables

Carrier solution (either water or DMSO) for the chemical inhibitors

controls

Positive controls: Not explicitly mentioned.
Negative controls: Carrier solution (either water or DMSO) for the chemical inhibitors.

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