BMC Cancer 2008, 8:30 http://www.biomedcentral.com/1471-2407/8/30 Wound Scratch Assay In order to investigate SK-N-SH cell migration capability after RA treatment, a modified wound scratch assay was used [19,24]. Briefly, the treated and untreated cells were grown to confluent monolayer on 13 mm diameter glass coverslips. On the 6th day of treatment, approaching 100% cell confluence, the monolayers were wounded by scratching the surface as uniformly as possible with a pipette tip. This initial wounding and the movement of the cells in the scratched area was photographically mon- itored using the Axiovert Zeiss 200 microscope with a 10× (NA 0.25) objective linked to a Coolsnap Es CCD camera (Roper Scientific – Crisel Instr. Rome) for 24 hr. This time interval has been chosen because it is shorter than SK-N- SH doubling time. Four different fields from each sample were considered for quantitative estimation of the dis- tance between the borderlines and in each image four dif- ferent equidistant points were measured in order to better estimate the real width of the wounded area. The migra- tion rate is expressed as percentage of the control, and it was calculated as the proportion of the mean distance between both borderlines caused by scratching, to the dis- tance which remained cell-free after re-growing [24]. Two independent series of experiments were performed in quadruplicates. To evaluate the correlation between DCX protein expression and cell migration capability, at the end of the photographic monitoring, the cells were fixed in 4% PFA and immunostained with anti-DCX antibody. Cell invasion assay Cell invasion was determined by a modified microchem- otaxis assay [20] using cell culture 24-well inserts with the bottom sealed by a 8 μm pore polycarbonate-filter coated with Matrigel (Chemicon International, Inc.). Briefly, 2.5 × 105 cells in 250 μl of serum-free MEM were seeded into the inserts. The lower chambers were filled with MEM additioned of 10%FBS. Chambers were incubated at 37°C in a 5% CO2 atmosphere for 48 hr. The cells that did not migrate through the filter were scraped from the insides of the inserts. The cells migrated to the underside of the coated filter were fixed in 4% paraformaldehyde for 5 min, washed two times with PBS and then processed for DCX IF analysis. Nuclei were stained with DAPI (Sigma). Alternatively, the migrated cells were incubated with cell detachment buffer and dissociated from the filter. The cells were then lysed and stained by CyQuant GR dye. Flu- orescence was counted using VICTOR3™ multilabel read- ers (PerkinElmer®). Data are expressed as means ± standard error, N = 4. Image acquisition Images were acquired using the UIC-Metavue 6.2.2 (UIC- Crisel Instr. Rome) imaging system on an Axiovert Zeiss 200 microscope. An oil immersion 63× (NA 1.4) or a 40× (NA 0.8) objective was used for IF images. To estimate the number of DCX-positive DCX+) or VIM-positive (VIM+) cells, at least 4 different images related to 3 control and 3 treated samples were considered. In each image acquired, the DCX+ and the VIM+ cells were counted. Data are graph- ically expressed as the ratio between the number of DCX+ or VIM+ cells to the number of the cells counted in total. For the wound scratch test analysis, images were acquired using a 10× (NA 0.25) objective. The distance between the borderlines of the scratched area was measured using the Region Measurement Function included in UIC-Metavue 6.2.2 (UIC-Crisel Instr. Rome) software. Statistical analysis Results of quantitative analysis are presented as means ± standard error (S.E.); the number of samples varied between a minimum of 4 and a maximum of 8 depending on the experimental protocol. Control and treated sam- ples have been compared by the one-sided unpaired Stu- dent's t-test or by ANOVA and using the GraphPad Prism software (version 4.0). The results are considered statisti- cally significant versus the untreated cells, with a proba- bility level of p < 0.05 (*), or statistically highly significant with a probability level of p < 0.01 (**). Results DCX is present in the N- and some I-type cells, but not in the S-type, of SK-N-SH cell line As already reported [2,3] the three different cell types composing SK-N-SH cell line were distinguishable by phase contrast (PC) microscopy analysis (Fig. 1A and 1F). IF analysis for DCX shows a clear signal (DCX+) in all the cells with the N morphological phenotype and in some I- type, while it is absent (DCX-) in the S-type cells (Fig. 1). In the cells that express DCX this protein is present in the cell body and all along the neuritic elongations, and, as expected due to its microtubule associated protein nature, it colocalizes with tubulin (Fig. 1). Since tumoral cells in culture may undergo genetic insta- bility the possible presence of DCX deleterious mutations was considered and ruled out by direct sequence analysis (see Materials and Methods) of the SK-N-SH DCX gene coding regions (data not shown). Since DCX can form etherodimers with LIS1, we also eval- uated LIS1 expression and we found its immunoreactivity in all the SK-N-SH cell subtypes, with a stronger signal in DCX+ cells (Fig. 1). To further characterize the cell subtypes not only on the basis of DCX expression, we considered other markers, such as VIM and NF68. 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