Sorted EpCAM+CD44+ cells were washed twice and diluted in 200 µl cold PBS containing 2% FBS. Slides and filters were placed into appropriate slots in a cytospin chamber (Stat Spin; Beckman Coulter, Tokyo, Japan) with the cardboard filters facing the center. In the event of few cells being available, 100 µl cold PBS containing 2% FBS was first placed in each cytospin, which was then spun at 250 × g for 5 min at 25°C to pre-wet the filter, allowing more cells to reach the slide. In addition, correct alignment of the filter/slide interface was ensured. For each sample, 200 µl was added to the appropriate wells of the cytospin, lids were applied and centrifugation was performed at 250 × g for 5 min at 25°C. Subsequently, the filters were removed taking care not to disturb the smears on the slides.
Each slide was examined under a microscope to check cell adherence, morphology and monolayer formation. Slides were dried overnight in a desiccator and evaluated using a transmitted light microscope (BX61/DP70; Olympus, Tokyo, Japan) equipped with an ultraviolet light source and filters. A cytotechnologist at the hospital analyzed the sorted cells with regard to the nuclear to cytoplasmic ratio, the overall cell size and the size of the nucleolus.
Each slide was examined under a microscope to check cell adherence, morphology and monolayer formation. Slides were dried overnight in a desiccator and evaluated using a transmitted light microscope (BX61/DP70; Olympus, Tokyo, Japan) equipped with an ultraviolet light source and filters. A cytotechnologist at the hospital analyzed the sorted cells with regard to the nuclear to cytoplasmic ratio, the overall cell size and the size of the nucleolus.