Three step stain set diff quik

Huh7^CD133+ and Huh7^CD133− cells were grown to 70% confluence in two 60-mm plates prior to experimentation for 12 h in 10% FBS after radiation treatment. Additionally, cells were washed and detached using 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS. After complete detachment, cells were resuspended in serum-free medium. Migration of Huh7^CD133+ and Huh7^CD133− cells was determined using a 24-well Transwell chamber system (Costar 3422, Corning Inc., NY, USA). Huh7^CD133+ and Huh7^CD133+ cells were seeded in the upper chamber at 3 × 10⁴ / mL in 0.2 mL serum-free DMEM medium. Medium supplemented with 10% fetal bovine serum was placed in the bottom well in a volume of 0.8 ml. After incubation for 12, 24 and 48 h, non-migrating cells on the upper side of the filter were removed with a cotton swab. The filters were stained with the Three-Step Stain Set (Diff-Quik; Sysmex, Kobe, Japan), and the cells that migrated to the lower side of the filter were counted under a slide scanner (Pannoramic MIDI; 3DHISTECH Ltd., Budapest, Hungary) in five randomly selected fields (× 200).