Kryomax colcemid

Murine Abelson virus-transformed B cells were generated by isolating total bone marrow from < 5 weeks old Atm^+/+Atm^−/− Pand Atm^RX/RX mice and infecting them with a retrovirus encoding the v-abl kinase (12 (link)). Cells were cultured in DMEM (Gibco, 12430–054) supplemented with 15% fetal bovine serum (Hyclone, SH30071.03), 1x MEM non-essential amino acids (Gibco, 11140–050), 2 mM L-glutamine (Gibco, 25030–081), 1 mM sodium pyruvate (Gibco, 11360–070), 1x penicillin/streptomycin (Gibco, 15140122) and 120 μM 2-mercaptoethanol (Fisher, 034461–100) for the next 6–8 weeks to allow for the generation of the stable clones. For treatment with Tert-Butyl Hydroperoxide (TBH) solution, cells were incubated in media without 2-mercaptoethanol. The proliferation of primary B cells was analyzed using the Cell Trace Violet (CTV) kit (ThermoFisher Scientific, C34557) according to the manufacturer’s protocol. Cells were analyzed by flow cytometry on an Attune NxT flow cytometer (ThermoFisher Scientific). The following small molecules were used at the concentrations indicated in the figure legends: TBH solution (Luperox TBH70X, Sigma, 458139), Colcemid (KryoMAX™ Colcemid™, Gibco, 15212–012), Neocarcinostatin (NCS) (Sigma, N9162).

Murine embryonic fibroblasts (MEFs) were harvested at embryonic day 14.5 and cultured in DMEM (Gibco, 12430-054) with 15% fetal bovine serum. Tert-Butyl Hydroperoxide (TBH), cells treated cells were incubated in 2-mercaptoethanol free medium. SV40 antigens immortalized the MEFs (33 (link)). MEF proliferation was measured by CellTiter Glo (Promega, G7572, 2×10³ cells/well on 96 wells) on a GloMax microplate reader (Promega). The relative growth was plotted as the fold increase over the first day [noted as day 0]. B cell proliferation was analyzed using the Cell Trace Violet (CTV) (ThermoFisher Scientific, C34557) and collected on an LSRII flow cytometer (BD Biosciences). Following chemicals were used: Camptothecin (CPT) (Calbiochem, 208925), Etoposide (EtOP) (Sigma, E1383), TBH solution (Luperox TBH70X, Sigma, 458139), Colcemid (KryoMAX^™ Colcemid^™, Gibco, 15212-012), and Neocarcinostatin (NCS) (Sigma, N9162).