Igh break apart probe

PDOX tumor cells were dissociated using collagenase, and then cultured in RPMI medium (GIBCO, Thermo Fisher Scientific) with 20% fetal bovine serum (FBS; GIBCO, Thermo Fisher Scientific), insulin (2.5 UI/ml) and 1× streptomycin and penicillin (GIBCO, Thermo Fisher Scientific) for 4 days. Conventional cytogenetic analysis was performed on cultured cells and chromosome G-banding was carried out using standard techniques. Interphase FISH was performed on fixed cells from bone marrow at diagnosis and from dissociated and cultured tumor cells with the following probes: XL IGH Break Apart Probe; XL t(4;14) FGFR3/IGH Translocation/Dual Fusion Probe; and XL t(14;16) IGH/MAF Translocation/Dual Fusion Probe (MetaSystems).

For conventional cytogenetic analysis, BM specimens were cultured for 24 and 72 h without mitogens, and stained by G-banding, following the standard techniques. In most of the cases, at least 20 metaphases were analyzed using an IKAROS imaging system (Metasystems, Altlussheim, Germany).
Interphase FISH (iFISH) analysis was performed using IgH Breakapart probe (Metasystems, Altlussheim, Germany), according to the manufacturer’s instructions. A minimum of 200 interphase nuclei and 10 metaphases were analyzed using a fluorescence microscope (AxioImager.Z1 mot, Carl Zeiss Ltd., Hertfordshir, UK) with ISIS imaging system (Metasystems, Altlussheim, Germany). The karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN 2016) [28 ].