Colony Formation Assay for Hematopoietic Cells

Cells were plated in a methylcellulose-based medium (Methocult H4230, Stemcell Technologies) and mixed in RPMI-1640 medium at a ratio of 1:4 (v/v) [67 (link), 68 (link)]. Harvested cells were mixed in a 1:10 (v/v) ratio with methylcellulose in 15 mL conical tubes that were then inverted gently. Thereafter, the contents were poured into 24-well plates, and incubated at 37°C in a 5% CO₂ incubator for 5 days. Then, p-iodonitrotetrazolium violet was added and incubated overnight. Colonies were visualized using the FluorChem 8800 imaging system (Alpha Innotech, San Leandro, CA).

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Yang L., Zhang S., George S.K., Teng R., You X., Xu M., Liu H., Sun X., Amin H.M, & Shi W. (2015). Targeting Notch1 and proteasome as an effective strategy to suppress T-cell lymphoproliferative neoplasms. Oncotarget, 6(17), 14953-14969.

Publication 2015

Methocult H4230 FluorChem 8800

Cells Iodonitrotetrazolium violet Methylcellulose Stemcell

Corresponding Organization :

Other organizations : Nantong University, Affiliated Hospital of Nantong University, The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Ratio of cells to methylcellulose-based medium (1:4 v/v)

dependent variables

Colony formation

control variables

Incubation at 37°C in a 5% CO2 incubator for 5 days
Addition of p-iodonitrotetrazolium violet and overnight incubation
Visualization of colonies using the FluorChem 8800 imaging system

controls

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

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