B cells were obtained either from Peyer’s patches of naïve mice or from the spleens of mice immunized with 100 μg NP(30 (link))-CGG (4-hydroxy-3-nitrophenylacetyl hapten, ratio = 30, chicken gamma globulin; Biosearch) in CFA for 2-4 weeks. Germinal center B cells were detected by staining with FITC-labeled anti-B220 (clone RA3-6B2) and Alexafluor647-labeled anti-GL7 (clone GL-7) antibodies (eBioscience). B220+GL7+ cells were isolated via flow cytometry, and DNA was prepared and amplified by nested PCR using JH4 intron primers as previously described (15 (link)). The PCR products were cloned and sequenced; only unique mutations were counted. Mutation spectra percentages were corrected for base composition of the amplified sequence.
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Mutation Analysis of Germinal Center B Cells
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4 hydroxy 3 nitrophenylacetyl
Antibodies
B cells
Cells
Chicken
Fitc
Flow cytometry
Gamma globulin
Germinal center
Hapten
Intron
Mice
Mutation
Nested pcr
Peyer patches
Primers
Corresponding Organization :
Other organizations : MRC Laboratory of Molecular Biology, National Human Genome Research Institute, National Institutes of Health
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Variable analysis
independent variables
- Immunization with 100 μg NP(30)-CGG in CFA for 2-4 weeks
- Germinal center B cells detected by staining with FITC-labeled anti-B220 and Alexafluor647-labeled anti-GL7 antibodies
- Unique mutations in the JH4 intron region of germinal center B cells
- Peyer's patches of naïve mice
- Positive control: Spleens of mice immunized with 100 μg NP(30)-CGG in CFA for 2-4 weeks
- Negative control: Peyer's patches of naïve mice
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