Gently pour off the medium and gently rinse with saline to remove protein etc, contained in the culture medium. Fix the colonies by overlaying them with sufficient 100% methanol (ethanol will also work) by adding to the dish or flask to completely cover the colonies - fix for 5 minutes or longer. Discard the methanol/ethanol, and add sufficient Crystal violet or Giemsa stain to cover the dish; wait 5 minutes, poor off and rinse with saline. I don't recall the exact concentration of crystal V or Giemsa (probably prepared in an aqueous alcohol solution), but tissue culture or general lab supply catalogues lists the concentration of the reagents. If you want to simply count colonies, Crystal V. works fine.
The following protocol has been modified from a published version (Franken et al., 2006).
Materials and Reagents
Cell culture medium
Phosphate buffered saline (PBS)
Fetal bovine serum (FBS)
Trypsin/ EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056)
Crystal violet (Sigma-Aldrich, catalog number: C3886)
Methanol (Sigma-Aldrich, catalog number: 34860)
Glacial acetic acid (Sigma-Aldrich, catalog number: 320099)
Fixation solution
Colony fixation solution (see Recipes)
Crystal violet solution (see Recipes)
Equipment
Cell culture petri dishes or six-well plates (Thermo Fisher Scientific, catalog number: 08-772-1B)
Hemocytometer (Hausser Bright-Line) (Thermo Fisher Scientific, catalog number: 02-671-10)
Stereomicroscope (e.g., Nikon Eclipse, model: TS100)
Hemocytometer
Incubator
Procedure
Cell preparation:Culture the cells according to the requirement (e.g., GBM cell lines, U87, U251, SF188, etc).
Remove medium, and then rinse cells with 10 ml PBS.
Add 4 ml 0.25% trypsin to the cells and incubate at 37 °C for 1-5 min until the cells appear round.
Add 10 ml medium with 10% FBS, and detach the cells by pipetting.
Count the cells using a hemocytometer.
Note: It is critical to get a relatively accurate number for the cells.
Prepare desired seeding concentration, and then seed cell into dishes or 6-well plates.
Assay setup:
Cell can be plated either before or after the treatment.Plating before treatment:Harvest cells and plate an appropriate number of cells per dish or per well on a 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 °C and allow them to attach to the plate/dish.
Treat the cells as necessary with chemicals (e.g., 1-100 μM), radiation (e.g., 2-10 Gy) or a combination of both.
Incubate the cells in a CO2 incubator at 37 °C for 1-3 weeks until cells in control plates have formed colonies that are of a substantially good size (50 cells per colony is the minimum for scoring).
Plating after treatment:Harvest cells after treatment. Fifty or up to 50 x 104 cells can be plated. Prepare serial dilutions with different numbers of cells, should the effects of the treatments be 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.
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).
Fixation and staining:Remove medium, and then rinse cells with 10 ml PBS.
Remove PBS and add 2-3 ml of fixation solution and leave the dishes/plates at room temperature (RT) for 5 min.
Remove fixation solution.
Add 0.5% crystal violet solution and incubate at RT for 2 h.
Add 10 ml medium with 10% FBS, and detach the cells by pipetting.
Remove crystal violet carefully and immerse the dishes/plates in tap water to rinse off crystal violet.
Air-dry the dishes/plates on a table cloth at RT for up to a few days.
Data analysis:Count number of colonies with a stereomicroscope.
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
Recipes
Colony fixation solution
Acetic acid/methanol 1:7 (vol/vol)
Crystal violet 0.5% solution
There are various methods to fix and stain colonies.
Provided below is one of example protocols that have been used in our studies.
Once colonies are formed (e.g., at 10-14 days after plating depending on cell lines), decant culture sup, wash once with 10 ml/4 dishes of Mg2+ - and Ca2+ free phosphate buffered saline [PBS(-)], fix with 5 ml/4 dishes of methanol (MeOH), and air-dry for >30 min
Add 10 ml/dish of crystal violet (CV) solution and stain for >3 hr at rt
Decant staining solution and air-dry.
CV solution consists of 0.0018% crystal violet, 0.015% formalin in tap water.
This protocol was used in the attached papers.
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There are various methods to fix and stain colonies.
Provided below is one of example protocols that have been used in our studies.
Once colonies are formed (e.g., at 10-14 days after plating depending on cell lines), decant culture sup, wash once with 10 ml/4 dishes of Mg2+ - and Ca2+ free phosphate buffered saline [PBS(-)], fix with 5 ml/4 dishes of methanol (MeOH), and air-dry for >30 min
Add 5 ml/dish of 5% Giemsa solution and stain for 30 min at room temperature
Decant staining solution, wash with tap water, and air-dry.
This protocol was used in the attached paper.
Article Gap junctional intercellular communication and cellular resp...
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