Department of Medicine, Case Western Reserve University, University Hospitals of Cleveland, 2103 Cornell road, Wolstein Research Building- 3rd Floor, Mailstop 3101, Cleveland, OH, 44106, USA,
Cytotechnology. 2007 Jun;54(2):85-8. doi: 10.1007/s10616-007-9083-2. Epub 2007 Jun 12.
Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.
集落形成实验是一种广泛应用的实验方法，用于测试药物/基因对细胞体外生长和增殖特性的影响。在集落形成实验中，准确地定量处理效果取决于精确观察和计数细胞集落的能力。我们报告了一种用于在塑料和特别涂覆的基质（如胶原）上生长的细胞集落染色的新方法（称为 ETeB）。使用在塑料和胶原上生长的结肠癌细胞系，我们比较了广泛使用的亚甲蓝和溴化乙锭（ETeB）染色剂的集落染色效率。结果表明，ETeB 方案在塑料上效果良好，与亚甲蓝相比，对胶原上的集落染色非常有效。ETeB 技术的关键特点和优势是：（a）减少在胶原和可能其他基质上生长的集落的背景，（b）整个过程耗时不到一分钟，（c）不需要进行染色后清洗步骤，从而避免了对松散附着的细胞系的集落损失，（d）染色后可立即使用标准 UV 照明器和软件进行集落可视化和计数，（e）该方法适用于广泛的细胞系。该方法的简单性和稳健性应该保证其在小规模和大规模集落形成实验中的使用。
