FFIiSgHuruesi3ng whole chromosome paint for chromosome 22 FISH using whole chromosome paint for chromo- some 22. Whole chromosome paint for chromosome 22 labeled with SpectrumOrange™, showing the presence of chromosome 22 fragments in (A) cell line ME and (B) cell line MER. FISH were denatured in 70% deionized formamide at 73°C for 5 min, dehydrated, and hybridized with 10 µ l hybridization mix consisting of labeled BAC probes in LSI hybridization buffer (Vysis) at 37°C for approximately 20 hours in a Perkin Elmer GeneAmp System 1000 in situ hybridization instrument. Slides were then washed in 0.4 × SSC (Vysis) for 2 min at 73°C, rinsed with 2 × SSC at room temperature, and left to air-dry. Preparations were counterstained with propiduim iodide or DAPI, and FISH signals were visualized and captured with a Zeiss Pascal 5 Laser Scanning Confocal Microscope. At least 75 met- aphase cells per cell line were examined; chromosome fragments were considered over-represented if three or more copies were seen per cell. Statistical analysis was done using the SAS (Statistical Analysis Software, SAS Institute, Inc, Cary, NC) PROC logistic regression method. Results Karyotype analysis Figure 2A and 2B show representative karyotypes from the primary (A) and recurrent (B) tumors from patient coded LX/LXR and Table 2 gives a summary of the results for all three patients. Karyotype analysis was done on low pas- sage cells to avoid artifacts that could arise from long term in vitro cultivation. Primary (passage 0) cells from tumors DI, DIR, ME and LX were prepared and analyzed as described in the Material and Methods. Cells from MER and LXR were analyzed following one serial passage. Pri- mary untreated tumors generally contained 2 or more non-related stem line populations which frequently dif- fered in the gain and/or loss of whole chromosomes in addition to different clonal markers. In contrast, recurrent tumor samples obtained within a year or less of the pri- mary surgery generally had only a single stem line in the samples we could analyze. The recurrent tumor stem lines also contain very complex karyotypes with multiple abnormalities including non-reciprocal translocations, deletions, duplications and marker chromosomes. Whole chromosomes 7 and/or 22 were over-represented in tumors DI and DIR [10]. In tumors ME and MER we were able to identify at least 2 normal and/or derivative chro- mosome 7 and 22's in addition to 1–5 marker chromo- somes. Chromosome 7 was over-represented in the primary tumor LX while in recurrent tumor LXR only one normal chromosome 7 was present along with 1–3 deriv- ative 7 s. Chromosome 22 was under-represented in the primary and over-represented in the recurrent tumor in addition to fragments within some of the derivative chro- mosomes and marker chromosomes. BMC Cancer 2006, 6:2 http://www.biomedcentral.com/1471-2407/6/2 release dividing cells into the medium. Cells were pel- leted, swelled using 0.075 M KCl at 37°C for 17 min and 22 sec, and fixed with methanol/acetic acid (3:1). Cells were then dropped onto slides, rinsed with methanol/ace- tic acid, dried, and stored until use [14]. Slides used for FISH analyses To identify the regions of chromosome 22 that were retained in the chromosome 22 fragments in cells from recurrent tumor and cells selected for BCNU resistance in vitro we used fluorescent in situ hybridization (FISH). We Page 5 of 10 (page number not for citation purposes)