Giemsa stain solution

The inhibitory effect of anchorage-independent cell growth after t-PDT was examined by soft agar colony-formation assays. Briefly, 2.5×10⁴ cells of TE-11R cells were suspended in 0.67% agarose containing media with or without talaporfin sodium (30 µg/mL), and overlaid on top of 1% agarose containing the medium per well. Subsequently, the gel was laser irradiated 24 h after the gel formation, and cells were grown for 2 weeks. Colonies were stained with 0.02% Giemsa Stain Solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), and the number and the size of colonies per high-power field were measured using a Nikon Eclipse TE300 microscope.

Cell migration assay was performed as previously described [56 (link)]. Briefly, cell culture inserts with polyethylene terephthalate (PET) filters containing 8.0 μm pores in the 24-well-plate format (BD Bioscience, Bedford, MA, USA) were used for the assay. The lower surface of the membrane was pre-treated with 10 μg/mL fibronectin (Sigma) for 30 min. MDA-MB-231 cells transfected with siTRIM44 or siControl (10 nM) 24 h before the assay were added to the upper chambers, and allowed to migrate for another 24 h. Then, the filters were dipped in methanol for 30 min, washed with PBS and stained with Giemsa stain solution (Muto Pure Chemicals, Tokyo, Japan) for 30 s. After washing 3 times with PBS, filters were mounted on glass slides. The cells migrated on the lower surface were counted in five randomly selected fields under a microscope at a magnification of ×400. Data are presented as mean value ± SEM.