Figure 2

Genetic analysis on BT1, BT1-neurospheres and adherent cells and BT1-tumors in nude mice. DNA was extracted from frozen tissues, cell cultures or lymphocytes, using standard protocols. Primers, microsatellite markers and PCR conditions for LOH analysis were described before [12]. We also investigated markers 9S157 and 9S171 flanking the CDKN2A gene on 9p21. Before doing microsatellite analysis on mouse tumors we confirmed that PCR primers did not hybridize on mouse DNA. For cytogenetic analysis cells were harvested with 0.1 μg/ml Colcemid (Karyomax Colcemid, Life Technologies) overnight. Hypotonic treatment, fixation and GTG banding of metaphase chromosomes were performed with standard methods. The karyotypes were described in accordance with ISCN guidelines http://cgap.nci.nih.gov/Chromosomes/Mitelman Spectral karyotyping was performed on metaphase cells according to the manufacturer's instructions (ASI, Carlsbad, CA) and to published procedures [13]. Spectral images were acquired and analyzed with an SD200 Spectral Bio-imaging System (ASI Ltd., MigdalHaemek, Israel) and a charged-coupled device camera (Hamamatsu, Bridgewater, NJ) connected to a Zeiss Axioskop 2 microscope (Carl Zeiss, Canada) and analyzed by the use of SKYVIEW (version 1.6.1; ASI) software. The upper panel shows the results of LOH analysis on 9p and 10q of the different samples outlined on the left. The lower panel illustrates a representative spectral karyotype of neurospheres obtained with the simultaneous hybridization of 24 combinatorially labeled chromosome painting probes. Karyotype display of chromosome banding (inverted DAPI) and SKY analysis (chromosomes were assigned a pseudo-color according to the measured spectrum) are shown. The number (7) next to the marker chromosome (der(3)) indicates the origin of inserted material.