Colony Assay for Hematopoietic Progenitors

Colony assays were performed in triplicate in 24 well plates using 1.2% methylcellulose (Dow Chemical, Waterloo, NY), 30% FCS, 57.2 μM β-mercaptoethanol, 2 mM glutamine, 0.5 mM hemin (Sigma, USA), 20 ng/mL interleukin 3 (IL-3), granulocyte colony stimulating factor (G-CSF), c-KIT ligand (KL) and 6 U/mL erythropoietin (Epo). IL-3 and KL and Epo were obtained from R and D Systems (Minneapolis, USA) and G-CSF was from Amgen (Thousand Oaks, CA). Colonies were scored 14 days after plating as per the protocol described earlier [42 (link)].

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Ambati S.R., Shieh J.H., Pera B., Lopes E.C., Chaudhry A., Wong E.W., Saxena A., Su T.L, & Moore M.A. (2016). BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models. Oncotarget, 7(28), 43062-43075.

Publication 2016

methylcellulose hemin G-CSF IL-3

Assays Erythropoietin Glutamine Granulocyte colony stimulating factor Hemin Interleukin 3 Kit ligand Mercaptoethanol Methylcellulose Oaks

Corresponding Organization : Memorial Sloan Kettering Cancer Center

Other organizations : Institute of Biomedical Sciences, Academia Sinica

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Variable analysis

independent variables

Methylcellulose concentration (1.2%)
FCS concentration (30%)
β-mercaptoethanol concentration (57.2 μM)
Glutamine concentration (2 mM)
Hemin concentration (0.5 mM)
Interleukin 3 (IL-3) concentration (20 ng/mL)
Granulocyte colony stimulating factor (G-CSF) concentration (20 ng/mL)
C-KIT ligand (KL) concentration (20 ng/mL)
Erythropoietin (Epo) concentration (6 U/mL)

dependent variables

Number of colonies scored 14 days after plating

control variables

Cell culture plates (24-well)
Plating in triplicate

controls

No positive or negative controls were explicitly mentioned.

Annotations

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