The culture dishes were harvested, and then the number of viable cells in each dish was counted by the dye exclusion test (0.1% trypan blue in PBS) GSK458 manufacturer every 24 hours for 7 days. Tumorigenicity in severe combined immunodeficiency (SCID) mice To determine the tumorigenicity of the FU-MFH-2 cell line in vivo, 5 × 107 cells at passage 23 were washed, suspended in PBS, and injected subcutaneously into the back of two 5-week-old female athymic SCID mice (CB-17/Icr-scid; Jcl Clea Japan, Inc., Osaka, Japan). The mice were maintained in a pathogen-free environment and carefully observed after transplantation. The experimental protocol was approved by the Ethics

Review Committee for Animal Experimentation of Fukuoka University Faculty of Medicine. Pathologic studies The cells grown in culture flasks were observed LY294002 solubility dmso by phase-contrast microscopy. FU-MFH-2 cells at passages 31 and 42 were examined. For routine light microscopy, the cells cultured in chamber slides (Lab-Tek, Miles Laboratories, Naperville, IL, USA) were fixed in methanol and stained with hematoxylin and eosin (H&E) and Giemsa. Paraffin sections from the original tumor and xenografts were stained with the same reagents. The primary antibodies and their dilutions used for

immunocytochemistry are listed in Table 1. The cells grown in chamber slides were washed in PBS and fixed in cold acetone for 5 minutes. The cells were reacted with each of the primary

antibodies for 1 hour at room temperature. The bound antibodies were then visualized using a labeled streptavidin biotin system and the alkaline phosphatase technique, as described previously [15]. Paraffin sections from the original tumor and xenografts were also examined using the same procedure. Table 1 Antibodies used in the present study. Antibody Type Source Dilution Vimentin M Dakopatts, Kyoto, Japan 1:50 EMA M Dakopatts 1:50 AE1/AE3 M Dakopatts 1:50 CAM 5.2 M Becton Dickinson, San Jose, CA, USA 1:50 Desmin M Dakopatts 1:50 α-SMA M Dakopatts 1:50 MSA (HHF35) M Enzo Diagnostics, Farmingdale, NY, USA 1:50 S-100 protein P Dakopatts 1:1000 NSE M Dakopatts 1:200 CD68 (KP-1) M Dakopatts 1:200 Lysozyme P Dakopatts 1:500 AAT P Dakopatts 1:1000 ACT P Dakopatts 1:1000 C-Kit P Immuno-Biological Laboratories, Fujioka, Thiamine-diphosphate kinase Japan 1:10 Abbreviations: EMA, epithelial membrane antigen; α-SMA, alpha-smooth muscle actin; MSA, muscle-specific actin; NSE, neuron-specific enolase; AAT, alpha-1-antitrypsin; ACT, alpha-1-antichymotrypsin; M, monoclonal (mouse); P, polyclonal (rabbit). Cytogenetic AZD1152 analysis The FU-MFH-2 cells at passages 25 and 52 and the fresh original tumor cells were used for cytogenetic analysis. Metaphase cells were banded with Giemsa trypsin, and karyotypic descriptions were done according to the International System for Human Cytogenetic Nomenclature 2009 [18].