Cao et al. BMC Cancer 2010, 10:68 http://www.biomedcentral.com/1471-2407/10/68 Cell culture NPC Cells were cultured in a 25-cm2 tissue culture flask (1.2 × 104cells/cm2) in RPMI 1640 supplemented with 10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin for 24 h. Cells were washed with PBS and the condi- tioned medium produced by PMA-activated THP-1 cells was added. After indicated cultivation days, nonadherent cells were gently washed away with PBS and adherent cells were harvested with 0.05% trypsin-EDTA for test. Flow cytometry To identify the SP cells in the cancer cells, we selected the single clone cells as described above. The cells were removed from the culture dish with trypsin/EDTA (Invi- trogen) and resuspended at 10 cells per ml RPMI 1640 supplemented with 2% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin. Cells were preincubated in a 1.5-ml Eppendorf tube for 90 min at 37°C with 2.5 μg/ml Hoechst 33342 dye (Invitrogen), either alone or in com- bination with 50 M verapamil, an inhibitor of some ver- apamil-sensitive ABC transporters [29]. Finally, the cells were counterstained with 1 μg/ml propidium iodide to label dead cells. Cells were analyzed in a FACSVantage fluorescence-activated cell sorter (Becton Dickinson). Propidium iodide-positive dead cells (<15%) were excluded from the analysis. The non-SP Cells from the single clone detected by FACS were seeded into a 6-well plate at 1 × 105 cells/ well and incubated with conditioned medium at 37°C. After 24 h of incubation, cells were collected with tryp- sin/EDTA. SK-N-CH cells were incubated with CD133- PE antibody for 10 minutes at 4°C before FACS analysis. CNE-2-2 single-cell suspension was fixed in ice-cold 70% ethanol at -20°C for 16 h, labeled with propidium iodide (50 μg/ml) for at least 15 min in dark and analyzed directly on a FACSCalibur flow cytometer (Becton Dick- inson Immunocytometry Systems, San Jose, CA, USA). Immunofluorescence staining Cells grown on cover slips were washed in cold PBS and fixed in 2% paraformaldehyde-PBS for 20 min and per- meabilized in 0.5% Triton X-100 in PBS for 10 min at 4°C. The cells were incubated with 1% BSA for 30 min at room temperature (RT) to block nonspecific binding before the primary antibody reaction. Cells were incubated with the primary antibodies to CD133 for 1 h at RT, and then a specific FITC conjugated secondary antibody for 1 h. Cells were counterstained with DAPI (1 μg/μl), and examined using a fluorescence microscope (Olympus BX51). Transwell migration assay CNE-2-2 cells were starved overnight in assay media (RPMI 1640 with 1% FBS). The top chamber of a 24- well transwell plate (Corning Inc.) was pretreated with Page 3 of 11 1% Matrigel™ (BD Biosciences, San Jose, CA, USA) in PBS and incubated 1 h at RT. Cells (105/ml) were added to the top chambers of the plates. Media with or with- out activated macrophage-like cells was added to both chambers. After overnight incubation, top cells were removed and bottom cells were fixed and stained with DAPI (5 μg/ml) to visualize nuclei. The number of migrating cells in five fields was counted under fluores- cence microscope, and the mean for each chamber was determined. Nucleic acid preparation All of the cells were snap-frozen in liquid nitrogen and stored at -80°C. The tested cells were lysed in Trizol Reagent (Invitrogen, CA, USA), and total RNA was iso- lated according to the manufacturer’s instructions. mRNA of the conditioned culture non-SP single cell clone and non-conditioned culture cells was purified from total RNA using the Oligotex mRNA Spin-Column kit (Qiagen, Hilden, Germany). Suppression subtractive hybridization (SSH) SSH was performed between conditioned culture non- SP single cell clone and non-conditioned culture cells using the PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA) according to the manufacturer’s recom- mendations. In the subtraction, conditioned culture non-SP single clone cells were used as a tester and non- conditioned culture single clone cells were used as a dri- ver. Double-strand cDNA was synthesized from poly (A)+ RNA and blunted according to the kit manual. Two tester populations were created with different adaptors, but no adaptors with the driver. After two cycles of hybridization, we carried out 28 cycles of primary PCR and 21 cycles of secondary PCR with DNA polymerase (Promega, Madison, WI, USA) on a GeneAmp 2400 thermal cycler (PE Biosystems, Foster City, CA, USA). To evaluate the efficiency of cDNA subtraction, we compared the expression of human b-actin (NP_001092) using the primers as denoted on Table 1 in subtracted and unsubtracted PCR products respectively. The subtracted fragments were then inserted into the T/A cloning vector pCR 2.1- TOPO (Invitrogen, CA, USA). Differential Screening 120 individual recombinant clones were picked and used as templates for PCR amplification using vector primers M13F and M13R. Each PCR product (about 1 μl) was transferred to a nylon membrane (Hybond-N, Amer- sham Pharamacia Biotech, Uppsala, Sweden). For dena- turing purposes, the membrane was submerged for 2 - 3 min in turn on the three pieces of Whatman 3 MM paper saturated with 1.5 M NaCl and 0.5 M NaOH, 1.5