Seed the cells
There are many published articles on how to perform colony formation assays. However, your specific cell line or experiment probably will require special adaptations (cell number, coating of the flask, culturing time, etc.). Therefore, you first have to optimize the conditions for your specific experiment.
There are some important factors that you should consider before performing the experiment to give you the best results with the CATCH-colonies software:
- You need a large amount of colonies (at least 1000 colonies per condition and biological replicate) to generate reliable data. Therefore, we recommend to use three T75 flasks per condition (approximately 200 cm² of growing area). Warning: Larger cell culture vessels (i.e. a T175 flask) may generate images that are too big for your computer to analyze.
- The colonies have to be clearly separated from each other. Therefore, we recommend to optimize the seeding density and incubation time so that you achieve a 1 % confluence when you stain the colonies. The easiest way to achieve this is to keep the incubation time as short as possible to prevent the colonies from overgrowing each other.
- To acquire high quality images on a flatbed scanner you have to overlay the colonies with a white contrast agent (i.e. starch powder). Therefore, we recommend to use cell culture flasks that can be tightly capped to keep the starch powder inside the flask. Using open cell culture plates with a loosely attached lid will cause the starch to spill everywhere.
Stain the colonies
When the colonies have reached the desired size, you have to stain the colonies for image acquisition. Many different protocols for this exist, but most commonly researchers use Crystal Violet. Be careful, Crystal Violet binds to DNA and is therefore suspected to cause cancer. Inform yourself about the safe usage and disposal of Crystal Violet.
- Remove the medium and wash the flasks once with PBS. If your cells do not attach sufficiently well to allow washing, it is possible to omit this step. This will cause proteins from the medium to form aggregates upon fixation, but typically the aggregates can be washed away after fixation.
- Fix the colonies with cold methanol (-20°C) for at least a few minutes.
- Remove the methanol and stain the cells with the crystal violet solution (0.5% Crystal Violet, 25 % methanol, diluted in water) for at least a few minutes. The staining solution can be re-used multiple times.
- Remove the staining solution and wash the flasks with water until all background staining is gone. Insufficient washing will lead lead to a spotty background (from dried in water droplets) that will confuse the software. Typically this will require up to 10 washing rounds to sufficiently reduce the background staining.
- Dry the flasks over night at room temperature. Try to remove all water droplets from the cell layer by flicking the flask.
Scan the colonies
Scanning a cell culture flask without contrast agent will lead to reflections from the side of the flask and a very poor result. To increase the contrast and avoid these reflections, fill the flask with pure white starch (i.e. from a supermarket). Compact the starch by tapping the flask until a homogeneous and tightly packed layer is formed on top of the colonies. Scan the colonies on a typical flat bed scanner in grayscale at a resolution of 4800 DPI with 8 bit. Make sure to adjust the contrast settings on the scanner to get high quality images.
Extract the colonies
- To start the program simply double-click the “CATCH-colonies” program for your operating system in the extracted ZIP folder.
- To create a new project simply click the “Create a new project” button in the main menu, then add the scans that you would like to analyse with the buttons on the left side.
- Using the buttons in the top bar you can go back an forth during the colony extraction step.
- Next, you have to select a region of interest (ROI) to exclude any unwanted regions in the scan. Simply click on the image to add ROI nodes and drag them around. The software will now convert the images to a special file format.
- The last step is to identify the colonies. The automatic mode uses a specially trained artificial intelligence to extract the colonies from the background. Can can choose between several settings for dense and loosely packed colonies. Use of the automatic mode is highly recommended. You also have to set a minimum colony diameter to exclude failed colonies that did not have unlimited replicative potential. Usually all colonies with less than 64 cells are excluded. Identify a few failed colonies under a microscope and then tweak the minimum colony diameter to exclude them.