Method Article

Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

April 30th, 2023

In This Article

Abstract

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Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).

This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

Protocol

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1. Colony-forming Unit Assays

  1. Thaw the methylcellulose with cytokines for mouse at room temperature for 1–2 h. Aliquot 3 mL of methylcellulose in a FACS tube using a 5 mL syringe without a needle. Sort the YFP-positive cells on a cell sorter.
  2. Spin the cells down and suspend the pellet in IMDM complete media. Count the cells to be plated and dilute them to obtain 5,000 YFP-positive cells in 100 µL of IMDM complete media.
  3. Add 100 µL of the cell suspension containing 5,000 YFP-positive cells and the appropriate inhibitors to 3 mL of methylcellulose. Vortex on high to mix it for 10–30 s.
  4. After most of the air....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
IMDMCellgro (corning)15-016-CVR
MethoCult GF M3434 (Methylcellulose for Mouse CFU)Stem Cell3434
4-HydroxytamoxifenSigmaH6278
Recombinant Murine SCFProspecCYT-275
Recombinant Murine Flt3-LigandProspecCYT-340
Recombinant Murine IL-6ProspecCYT-350
Recombinant Murine IL-7Peprotech217-17
DFCLKT Laboratories Inc.D3420
BCIChemzon ScientificNZ-06-195
ImatinibLC LaboratorI-5508
1x PBSCellgro (corning)21-040-CV
1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2Becton Dickinson329461
Iodonitrotetrazolium chlorideSigmaI10406
Instruments
NAPCO series 8000 WJ CO2 incubatorThermo scientific
Swing bucket rotor centrifuge 5810REppendorf
C-1000 Thermal cyclerBio-RAD
ChemiDoc Imaging System (imaging system for gels and western blots)Bio-RAD17001401
Mice
ROSACreERT2Jackson Laboratory
ROSACreERT2/c-Fos fl/fl Dusp1-/Made in house
ROSACreERT2/c-Fosfl/flMade in house

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Tags

Methyl Cellulose CFU AssayColony Formation UnitAnticancer CompoundsProgenitor CellsMethylcellulose MatrixIodonitrotetrazolium StainCytokine StimulationColony CountingCell Culture Incubation

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