Clonogenic Assay: Adherent Cells

1. Cell Culture and Experimental Set-up

1. Human keratinocytes are maintained as monolayers in 75 cm² tissue culture flasks containing 15 mL of keratinocyte-SFM (K-SFM) medium (GIBCO, serum-free medium) supplemented with L-glutamine (2 mM), epidermal growth factor (5 ng/ mL), bovine pituitary extract (40 μg/ mL) and 20 mg/ mL gentamicin. Cells are grown in a humidified 5% CO₂ environment at 37°C.
2. Cells are seeded into 12 x 25 cm² tissue culture flasks containing 5 mL of K-SFM medium.

Single cell suspensions are prepared by trypsinization. Cells are washed with phosphate buffered saline and incubated with a 0.05% trypsin / EDTA solution for 5-10 minutes. When the cells start to become rounded and ~30% are detached, 3 volumes of Dulbecco's modified eagle medium containing 10% fetal bovine serum is added to neutralize the trypsin. The cells are detached by pipetting up and down (20 times). Cells are counted using a hemocytometer.

Appropriate cell numbers are seeded according to the doubling time of the cell line (approximately 20 hours for human FEP-1811 keratinocytes). The aim is to achieve ~90% confluency (~10⁶ cells per flask) on the day of the experiment.

An experiment consisting of 12 flasks is optimal for a single clonogenic assay (six unirradiated control and six irradiated flasks) which can be completed in approximately four hours.

2. Treatment and Irradiation

1. Treat cells for an appropriate time with a relevant radiation-modifying compound and expose cells to ionising radiation either γ-radiation or X-rays.

Typically six flasks serve as plating efficiency (untreated) and drug only controls. The other six flasks are irradiated.

In this example human keratinocytes are treated with various concentration of Cinnulin PF (CPF; Integrity Nutraceuticals International, Spring Hill, TN, US; representative data for 20 μg/ mL is shown below), a water-soluble natural antioxidant formulation, for 1 hour at 37°C. Cells are irradiated with 4 Gy using a ¹³⁷Cs source (Gammacell 1000 Elite irradiator; Nordion International, ON, Canada; 1.6 Gy/min).

3. Plating

1. Following treatment, single cell suspensions are obtained as described earlier.
2. The number of cells in each sample are counted carefully using a hemocytometer and diluted such that appropriate cell numbers are seeded into petri dishes (five replicates of each in 15 mm dishes).
   The plating efficiency and / or surviving fraction should be anticipated when deciding the number of cells to seed per plate. The aim is to achieve a range of between 20 - 150 colonies.

Petri dishes are arranged in a humidified plastic cloning box and incubated in a 5% CO₂ environment at 37°C for colony formation.

The incubation time for colony formation varies from 1-3 weeks for different cell lines; it is accepted that the time must be equivalent to at least six cell divisions. In this example, the control dishes for human keratinocytes require eight days to form sufficiently large clones consisting of 50 or more cells.

4. Fixing and Staining Colonies

Complete the following steps in a fume hood.

1. Gently remove the media from each of the plates by aspiration.
2. Wash each plate with 5 mL 0.9% saline.
3. Fix the colonies with 5 mL 10% neutral buffered formalin solution for 15-30 minutes.
4. Stain with 5 mL 0.01% (w/v) crystal violet in dH₂O for 30-60 minutes.
5. Wash excess crystal violet with dH₂O and allow dishes to dry.

5. Colony Counting

Stereomicroscope

1. Colonies containing more than 50 individual cells are counted using a stereomicroscope.

Digital imaging and counting using imaging software

1. Digital images of the colonies are obtained using a camera or scanning device
2. Colonies are counted using imaging analysis software packages as described below.

Cell counting using ImageJ (Fiji Version 1.44a)

1. Open the image file in Fiji, go to File -> Open.
2. If required convert the image to 8-bit format, go to Image -> Adjust...-> Threshold.
3. Adjust threshold to reduce levels of non-specific background so that only the colonies are detected.
4. Count colonies using the following: go to Process -> Binary -> Find maxima.

For this image format, noise tolerance can be set to 0. Ensure that light background option is ticked and preview the detected maxima to check that all cell colonies have been correctly registered.

In this example human FEP-1811 keratinocytes were treated with various concentrations up to 100 μg/ mL CPF; data is shown for 20 μg/ mL CPF for 1 hour at 37°C. Following treatment cells were irradiated with 4 Gy using a ¹³⁷Cs source (Gammacell 1000 Elite irradiator; Nordion International, ON, Canada; 1.6 Gy/min). For the control untreated and drug only treatments 100 cells were plated in each petri dish and 1000 cells per dish were plated for the irradiated samples. As indicated in the table above, colonies were counted using a stereomicroscope or digital images (Fig 1) were taken for counting using ImageJ. The average colony count for the five dishes was used to calculate plating efficiency and surviving fraction. The data indicated a radiation protective effect (protection factor ~3) with the natural antioxidant formulation.

Representative Results

¹ Plating efficiency = number of colonies counted / number of cells plated
² Surviving fraction = (number of colonies counted /number of cells plated) / plating efficiency

Table 1. Calculation of plating efficiency and survival and comparison of colony counts using various methods

Figure 1. Digital image showing colonies produced by human, FEP-1811, keratinocytes following plating of 1000 cells and eight days incubation. (A) Cells were irradiated with 4 Gy and (B) cells were treated with 20 μg/ mL CPF for 1 hour at 37°C prior to irradiation with 4 Gy. A radiation protective effect with the natural antioxidant formulation is evident.