B Cell Isotype Switching Assay

B cells were stimulated with LPS as described above and with 0.5 μg/ml anti-mouse CD40 (clone FGK45, Enzo Life Sciences). The following cytokines were then added for specific switching: for IgG1, IL-4 as described above; for IgG2b, 2 ng/ml TGF-β (R&D Systems); for IgG3, no additional cytokines; and for IgA, IL-4, TGF-β, and 1.5 ng/mL mouse IL-5 (R&D Systems). Flow cytometry analysis of switched populations was conducted after 4 days using cells stained with FITC- or PerCP-labeled anti-B220 (clone RA3-6B2, eBioscience), and either allophycocyanin-conjugated anti-IgG1 (clone M1-14D12, eBioscience), recombinant PE-conjugated IgG2b, IgG3, or IgA antibodies (Southern Biotech). For germline transcripts, B cells were stimulated 4 days as described above. mRNA was harvested and converted to cDNA as stated for AID qPCR. PCR was performed as given previously (17 (link), 18 (link)) using primers synthesized by IDT.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Zanotti K.J., Maul R.W., Castiblanco D.P., Yang W., Choi Y.J., Fox J.T., Myung K., Saribasak H, & Gearhart P.J. (2014). ATAD5 Deficiency Decreases B Cell Division and Igh Recombination. Journal of immunology (Baltimore, Md. : 1950), 194(1), 35-42.

Publication 2014

TGF-β allophycocyanin-conjugated anti-IgG1 (clone M1-14D12,

Allophycocyanin B cells Cdna Cells Clone Cytokines Fgk45 Fitc Flow cytometry Germline Iga antibodies Igg1 Igg2b Igg3 Mouse Mrna Populations Primers Tgf β

Corresponding Organization :

Other organizations : MRC Laboratory of Molecular Biology, National Human Genome Research Institute, National Institutes of Health

Top 5 similar protocols

Variable analysis

independent variables

LPS stimulation
Anti-mouse CD40 (clone FGK45, Enzo Life Sciences) at 0.5 μg/ml
Cytokine addition for specific switching: IL-4 for IgG1, 2 ng/ml TGF-β (R&D Systems) for IgG2b, no additional cytokines for IgG3, IL-4, TGF-β, and 1.5 ng/mL mouse IL-5 (R&D Systems) for IgA

dependent variables

Switched populations analyzed by flow cytometry after 4 days
Germline transcripts measured by PCR after 4 days of stimulation

control variables

Not explicitly mentioned

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!