A simple technique for isolating pure cell populations from mixed primary cultures

We describe a simple method for isolating designated cell colonies from mixed primary cultures. Circles surrounding the chosen colonies are drawn on the underside of a 60-mm tissue culture dish. The medium in the dish is drained except for about 0.5 ml. The dish is tilted and the area surrounding a marked colony to a distance of about 5 mm is scraped with a sharpened rubber policeman. The low fluid level in the dish facilitates accurate scraping, while the dish is frequently reverted to a horizontal position to spread the retained medium and prevent inadvertent drying. When scraping is completed, the surface of the dish outside the marked areas is dried to destroy cells that escaped scraping and to prevent trypsin from spreading outside the marked areas. Then, either medium can be replenished to allow further growth of the colonies in the same dish, or each cell colony can be individually trypsinized or scraped off the dish for transferral to another dish for subsequent passaging.