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.
Fitc labeled anti b220 clone ra3 6b2
Manufactured by Thermo Fisher Scientific
FITC-labeled anti-B220 (clone RA3-6B2) is a fluorescently labeled monoclonal antibody that binds to the B220 antigen, also known as CD45R, which is expressed on the surface of B cells. This product can be used in flow cytometry and other immunological applications to identify and quantify B cells in various samples.
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2 protocols using fitc labeled anti b220 clone ra3 6b2
1
Mutation Analysis of Germinal Center B Cells
2
Isolation and Analysis of Germinal Center B Cells
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Lymphocytes were isolated from Peyer’s patches and stained with FITC-labeled anti-B220 (clone RA3-6B2; eBioscience) and Alexa Fluor 647–labeled anti-GL7 (clone GL7; BioLegend) antibodies, followed by cell sorting for the B220+ GL7+ germinal center B cell population. Cells were then lysed in digestion buffer (10 mM Tris, pH 8.0, 25 mM EDTA, 100 mM NaCl, 1% SDS, and 0.1 mg/ml proteinase K) at 55°C overnight. Genomic DNA was isolated by phenol/chloroform extraction and ethanol precipitation. The JH4 intronic region was amplified and analyzed as previously described (Saribasak et al., 2012 (link)). For mutation frequency and spectra, mutations were analyzed from previously published data (2,866 mutations) from C57BL/6 mice (Rada et al., 2004 (link); Martomo et al., 2008 (link); Schenten et al., 2009 (link); Saribasak et al., 2011 (link); Zanotti et al., 2015 (link)). Poli129/129 data were from McDonald et al. (2003) (link) and data generated in this study, and Polim/m data were generated in-house.
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