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Clonogenic assay serves as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells which can often only be determined microscopically. Clonogenic assay is the method to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. The following protocol has been modified from a published version (Franken, Rodermond et al. 2006).
Materials and reagents
1. Cell culture medium
4. Trypsin/ EDTA (Invitrogen, 25200056)
5. Crystal violet (Sigma-Aldrich, C3886)
6. Methanol (Sigma-Aldrich, 34860)
7. Glacial acetic acid (Sigma-Aldrich, 320099)
1. Cell culture petri dishes or six-well plates (Fisher Scientific, 08-772-1B)
2. Hemocytometer (Hausser Bright-Line, Fisher Scientific, 02-671-10)
3. Stereomicroscope (e.g., Nikon Eclipse TS100)
1. Cell preparation:
1) Culture the cells according to the requirement (e.g., GBM cell lines, U87, U251, SF188, etc).
2) Remove medium, and then rinse cells with 10ml PBS.
3) Add 4ml 0.25% trypsin to the cells and incubate at 37 °C for 1-5min until the cells appeared round.
4) Add 10ml medium with 10% FBS, and detach the cells by pipetting.
5) Count the cells using hemocytometer.
Note: it is critical to get a relatively accurate number for the cells.
6) Prepare desired seeding concentration, and then seed cell into dishes or 6-well plates.
2. Assay Setup:
Cell can be plated either before or after the treatment.
1) Plating before treatment:
a. Harvest cells and plate an appropriate number of cells per dish or per well on 6-well plate, at least in duplicate. The number of cells for seeding should be determined by the aggressiveness of the treatment. Incubate cells for a few hours in a CO2 incubator at 37°Cand allow them to attach to the plate/dish.
b. Treat the cells as necessary with chemicals (e.g., 1-100 μM), radiation (e.g., 2-10 Gy) or combination of both.
c. Incubate the cells in a CO2 incubator at 37°C for 1-3 weeks until cells in control plates have formed colonies with substantially good size (50 cells per colony is the minimum for scoring).
2) Plating after treatment:
a. Harvest cells after treatment. 50 or up to 50x104 cells can be plated. Prepare serial dilutions with different numbers of cells, should the effects of the treatments are unclear. For radiation treatment, the cells can be plated immediately after treatment or re-plated later. It is always better to keep the cells on ice before re-plating.
b. Incubate the cells in a CO2 incubator at 37°C for 1-3 weeks until cells in control plates have formed colonies with substantially good size (50 cells per colony is the minimum for scoring).
3. Fixation and Staining:
1) Remove medium, and then rinse cells with 10ml PBS.
2) Remove PBS and add 2-3ml of fixation solution and leave the dishes/plates at room temperature for 5 min.
3) Remove fixation solution.
4) Add 0.5% crystal violet solution and incubate at room temperature for 2 hours.
5) Add 10ml medium with 10% FBS, and detach the cells by pipetting.
6) Remove crystal violet carefully and immerse the dishes/plates in tap water to rinse off crystal violet.
7) Air-dry the dishes/plates on a table cloth at room temperature for up to a few days.
4. Data analysis:
1) Count number of colonies with a stereomicroscope.
2) Calculate plating efficiency (PE) and surviving fraction (SF).
PE = no. of colonies formed/ no. of cells seeded X 100%
SF = no. of colonies formed after treatment/ no. of cells seeded X PE
1. Colony fixation solutions
Acetic acid/methanol 1:7 (vol/vol)
2. Crystal violet, 0.5% solution
1. Franken N.A., Rodermond H.M., Stap J., Haveman J., van Bree C. (2006). Clonogenic assay of cells in vitro. Nat Protoc 1(5): 2315-9.
2. Mueller S., Yang X., Sottero T.L., Gragg A., Prasad G., Polley M.Y., Weiss W.A., Matthay K.K., Davidoff A.M., DuBois S.G., Haas-Kogan D.A. (2011). Cooperation of the HDAC inhibitor vorinostat and radiation in metastatic neuroblastoma: efficacy and underlying mechanisms. Cancer Letters 306(2): 223-9.
3. Prasad G., Sottero T., Yang X., Mueller S., James C.D., Weiss W.A., Polley M.Y., Ozawa T., Berger M.S., Aftab D.T., Prados M.D., Haas-Kogan D.A. (2011). Inhibition of PI3K/mTOR pathways in glioblastoma and implications for combination therapy with temozolomide. Neuro Oncol 13(4): 384-92.
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