B cells were isolated from mice aged 5–7 wk. Splenic B cells were isolated using B220 magnetic beads (Miltenyi Biotec) or by FACS sorting. B220+IgM− cells were purified via FACS sorting from bone marrow. Genomic DNA samples were obtained using phenol-chloroform extraction of whole-cell lysates. HTGTS–Rep-seq libraries were prepared as previously described (Hu et al., 2016 (link); Lin et al., 2016 (link)). Briefly, genomic DNA was sonicated and subjected to linear amplification–mediated PCR using biotinylated JH1 or JH3 bait primers (Lin et al., 2016 (link)). Linear amplification–mediated PCR products were purified using Dynabeads MyONE C1 streptavidin beads (Life Technologies; 65002) and ligated to bridge adaptors. Adaptor-ligated products were amplified by nested PCR with indexed JH1/JH3 primers (8-bp indexes were unique to each sample; Table S9 ) and primer annealed to the adaptor. PCR products were further tagged with Illumina sequencing adaptor sequences and size-selected via gel extraction. Libraries were sequenced by paired-end 300-bp sequencing on an Illumina MiSeq by the Yale Center for Genome Analysis. Primers were previously described and are listed in Table S9 (Hu et al., 2016 (link); Lin et al., 2016 (link)).
B220 magnetic beads
Manufactured by Miltenyi Biotec
B220 magnetic beads are a type of laboratory equipment used for cell separation and isolation. They are small, uniform, superparamagnetic particles coated with an antibody specific for the B220 antigen, a cell surface marker found on B lymphocytes. When cells expressing the B220 antigen are exposed to the beads, they become magnetically labeled and can be separated from other cell types using a magnetic field.
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Lab products found in correlation
2 protocols using b220 magnetic beads
1
High-Throughput Genomic Profiling of B Cell Repertoire
2
Adoptive Transfer of P. yoelii Immune B Cells
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Splenic B cells of mice that had cleared P. yoelii infection 4 months earlier were isolated by using B220 magnetic beads (Miltenyi Biotec, San Diego, CA). The purity of B220+CD19+ B cells was > 95%. Plasmodium yoelii immune B-cells were then adoptively transferred by intravenous (i.v.) injection of 3 × 107 immune cells per mouse. Two hours after the B cell transfer, mice were injected with P. yoelii infected RBCs. The percent parasitemia (parasitized RBCs/total RBCs × 100) after infection was determined by examining Giemsa-stained thin blood smears.
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