Splenic B cell isolation and FISH analysis

Splenic B cells were isolated from naïve mice, purified by negative selection kit (Stem Cell Technologies, Canada), activated with anti-CD40 and IL4 as described previously [82 (link)], and collected 4 days after culture for metaphase preparation and FISH analysis. FISH analysis was performed with specific BAC probes as previously described [82 (link)] (see details in Additional file 1). Genomic DNA was isolated from tumor masses or normal tissues from control mice, and Southern blotting was performed as previously described [41 (link)].

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Chen Z., Elos M.T., Viboolsittiseri S.S., Gowan K., Leach S.M., Rice M., Eder M.D., Jones K, & Wang J.H. (2016). Combined deletion of Xrcc4 and Trp53 in mouse germinal center B cells leads to novel B cell lymphomas with clonal heterogeneity. Journal of Hematology & Oncology, 9, 2.

Publication 2016

negative selection kit

B cells Fish Genomic Metaphase Mice Normal tissues Splenic Stem cell Tumor

Corresponding Organization : University of Colorado Anschutz Medical Campus

Other organizations : University of Colorado Denver, National Jewish Health

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Isolation of splenic B cells from naïve mice
Purification of splenic B cells using negative selection kit
Activation of splenic B cells with anti-CD40 and IL4
Culture of activated splenic B cells for 4 days

dependent variables

Metaphase preparation
FISH analysis
Southern blotting of genomic DNA from tumor masses and normal tissues

control variables

Negative selection kit used for purification of splenic B cells
Culture conditions and duration (4 days) for activated splenic B cells
Procedures for FISH analysis and Southern blotting as previously described in references [82] and [41]

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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