Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells
Published: April 30, 2023
Abstract
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
1. Colony-forming Unit Assays 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. 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. Add 100 µL of the cell suspension containing 5,000 YFP…
Divulgaciones
The authors have nothing to disclose.
Materials
| IMDM | Cellgro (corning) | 15-016-CVR | |
| MethoCult GF M3434 (Methylcellulose for Mouse CFU) | Stem Cell | 3434 | |
| 4-Hydroxytamoxifen | Sigma | H6278 | |
| Recombinant Murine SCF | Prospec | CYT-275 | |
| Recombinant Murine Flt3-Ligand | Prospec | CYT-340 | |
| Recombinant Murine IL-6 | Prospec | CYT-350 | |
| Recombinant Murine IL-7 | Peprotech | 217-17 | |
| DFC | LKT Laboratories Inc. | D3420 | |
| BCI | Chemzon Scientific | NZ-06-195 | |
| Imatinib | LC Laborator | I-5508 | |
| 1x PBS | Cellgro (corning) | 21-040-CV | |
| 1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2 | Becton Dickinson | 329461 | |
| Iodonitrotetrazolium chloride | Sigma | I10406 | |
| Instruments | |||
| NAPCO series 8000 WJ CO2 incubator | Thermo scientific | ||
| Swing bucket rotor cetrifuge 5810R | Eppendorf | ||
| C-1000 Thermal cycler | Bio-RAD | ||
| ChemiDoc Imaging System (imaging system for gels and western blots) | Bio-RAD | 17001401 | |
| Mice | |||
| ROSACreERT2 | Jackson Laboratory | ||
| ROSACreERT2/c-Fos fl/fl Dusp1-/ | Made in house | ||
| ROSACreERT2/c-Fosfl/fl | Made in house |
Citar este artículo
Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells. J. Vis. Exp. (Pending Publication), e20277, doi: (2023).