#Imagej colony counting softwareThe software tested for colony size distribution analysis is divided into two parts: the macro which allows us to measure the size of the automatically (or semiautomatically) counted colonies and the program dedicated to draw distributions of colony sizes in the form of histograms. The program countPHICS is freely available at. It consists of a macro written for ImageJ which analyses computerised images of cell culture dishes and a program which plots histograms of colony size distribution and fits the best function. Such a tool was developed by us and is described herein. 2007) but they all focus on enumerating colonies and not on a systematic analysis of colony sizes. Thus, there is a need for an image analysis tool which would estimate the areas of colonies, best in an automated and high throughput setup that is supplemented by a statistical analysis of the results.Ī number of automated counting programs have been developed (Barber et al. The area of a colony can be estimated by measuring its diameter but this assumes a round shape which is often not given. Counting of cells in a colony can be carried out with an inverted microscope but this is time consuming. 2006), which corresponds to seven cell divisions assuming no cell death. Traditionally, a dense conglomerate of cells is regarded as a colony when the number of cells exceeds 50 (Franken et al. While the colony number can be quantified by eye, the analysis of colony size requires some kind of measurement. Later, it was suggested that the heritable lesions are a manifestation of genomic instability (Pampfer and Streffer 1989). This observation was confirmed and pursued by Beer (Beer 1979 Beer and Szumiel 1994). Already in 1964 Sinclair discovered that ionising radiation induces some heritable lesions which lead to colonies of small size (Sinclair 1964). Hence, cells have time to express phenotypic effects which require time and possibly several cell divisions for development. As compared to most assays which are used to assess the genotoxic or cytotoxic effects of various agents, the great advantage of CCSA is that it measures the ability of cells to retain their reproductive integrity after a prolonged period of time, usually between 1 and 2 weeks following exposure (Franken et al. 1967) and the variable radiosensitivity of cells in different phases of the cell cycle (Sinclair and Morton 1966). CCSA was instrumental in discovering such essential phenomena in radiation research as the dose rate effect (Hall and Bedford 1964), lethal and sublethal radiation damage (Elkind et al. 1956) following the discovery that cells grown under in vitro conditions require conditioned medium if they are to form colonies from a single precursor (Sanford et al. The clonogenic cell survival assay (CCSA) is a basic method used in radiation biology and toxicology to estimate the cytotoxic effect of physical or chemical toxins. In conclusion, our new publically available software will facilitate colony counting and provide additional information on the colony growth rate, which is relevant especially for radiosensitisation studies. The software called count and Plot HIstograms of Colony Size (countPHICS) consists of two parts: (1) a macro written for ImageJ which analyses computerised images of cell culture dishes or 6-well plates, counts colonies, estimates their size and saves the results in a text file (2) a program written with QT Creator which reads the text file, plots histograms of colony size distribution and fits the best function. We have developed a new software package for both counting colonies and plotting their size distributions. Although a number of such systems exist, they all focus on enumerating colonies and not on analysing the colony size. Such analyses are largely facilitated by computerised image analysis systems. Moreover, it is often interesting to quantify the size of individual colonies. In large experimental setups, counting of colonies by eye is tiresome and prone to bias. The clonogenic cell survival assay is a basic method to study the cytotoxic effect of radiation and chemical toxins.
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