Metaphase Analysis of Splenic B-cells

Splenic B-cells from 8–12 week old mice were purified with anti-CD43 MACS beads and stimulated with LPS/IL4 for 2–4 days as described previously (20 (link)). Metaphases were obtained at day 4.5 after stimulation and stained with Cy3 conjugated PNA probe against 3xtelomere sequence as previously described (25 ). Tumor metaphases were harvested after 6 hours incubation in RPMI1640 with15% Fetal Bovine Serum in the presence of colcemid (100ng/ml, Invitrogen, CA). Chromosome 12 paint was obtained from Applied Spectral Imaging (Carlsbad, CA) and the 5′ c-myc (C10) and 5′ IgH (BAC207) locus-specific FISH probes were described previously (26 (link)). Images were acquired with Zeiss Axio Imager Z2 equipped with a CoolCube1 camera (Carl Zeiss, Thornwood, NY) and an automatic stage system, and processed with Isis and Metafer4 software packages (MetaSystems, Newton, MA).

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Yamamoto K., Lee B.J., Li C., Dubois R.L., Hobeika E., Bhagat G, & Zha S. (2015). Early B-cell Specific Inactivation of ATM Synergizes with Ectopic CyclinD1 Expression to Promote Pre-germinal center B-cell Lymphomas in Mice. Leukemia, 29(6), 1414-1424.

Publication 2015

colcemid CoolCube1 camera Metafer4

B cells Cd43 Chromosome 12 Colcemid Fetal bovine serum Fish Metaphases Mice Splenic Tumor

Corresponding Organization :

Other organizations : Columbia University, University of Freiburg

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Variable analysis

independent variables

Stimulation with LPS/IL4 for 2-4 days

dependent variables

Metaphase chromosome structure and telomere signals
Chromosome 12 paint signals
5′ c-myc and 5′ IgH locus-specific FISH probe signals

control variables

Splenic B-cells from 8–12 week old mice
Purification of B-cells with anti-CD43 MACS beads
Incubation time of tumor metaphases (6 hours)
Culture medium (RPMI1640 with 15% Fetal Bovine Serum)
Colcemid concentration (100ng/ml)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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