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Methylcellulose colony assays

ES Emmanuelle Supper
SR Saskia Rudat
VI Vivek Iyer
AD Alastair Droop
KW Kim Wong
JS Jean-François Spinella
PT Patrick Thomas
GS Guy Sauvageau
DA David J. Adams
CW Chi C. Wong
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Murine bone marrow c-Kit+ cells were resuspended in IMDM media (Gibco) and added to M3434 methylcellulose media (STEMCELL Technologies) before being dispensed into 35 mm dishes to obtain 500 cells/dish. Cells were incubated at 37 °C in 5% CO2 for seven days before scoring colony numbers. Patient-derived hematopoietic and transduced human CD34+ cells were mixed with H4435 methylcellulose media (STEMCELL Technologies) and plated into 35 mm dishes with birinapant or drug vehicle as indicated (1-3 × 105 plate for patient-derived cells; 500 cells/plate for human CD34+ cells). Plates were incubated for 13 days before staining with 0.1% p-iodonitrotetrazolium violet solution overnight. Plate images were captured and colonies were enumerated using ImageJ software (v1.52b, NIH) or counted manually.

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