Lymphocyte Development and CSR Analysis

Lymphocyte development and in vitro CSR were performed as described before (10 (link),31 (link),32 ). Hematopoietic cells from 5–8 week mice were stained and analyzed on a FACS Calibur flow cytometer (BD Biosciences). The antibodies are: PE-CD4 (clone GK1.5, BD Pharmingen, 553730), FITC-CD8α (clone 53–6.7, BioLegend, 100705), APC-TCRβ (clone H57-597, BD Pharmingen, 553174), PE/Cy7 TER-119 (clone TER-119, BioLegend, 116222), FITC-CD43 (clone S7, BD Pharmingen, 553270), PE-Cy5-B220 (clone RA3-6B2, BD Pharmingen, 553091) and PE-IgM (Southern Biotech, 1020-09). For in vitro CSR, CD43⁻ splenic cells (anti-CD43 magnetic beads, Miltenyi, 130-049-801) were cultured (~1 × 10⁶ cells ml⁻¹) in RPMI (Gibco, 11875-093), serum supplements (10 (link),31 (link),32 ), IL-4 (20 ng/mL; R&D, 404-ML-050), and anti-CD40 (1 μg/mL; BD Bioscience, 553721) and analyses with FITC-conjugated IgG1 (clone A85-1, BD Pharmingen, 553443) and PECy5-conjugated B220 (clone RA3-6B2, BD Pharmingen, 553091). FlowJo software package was used for data analyses

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.

Milanovic M., Houghton L.M., Menolfi D., Lee J.H., Yamamoto K., Li Y., Lee B.J., Xu J., Estes V.M., Wang D., Mckinnon P.J., Paull T.T, & Zha S. (2020). The cancer-associated ATM R3008H mutation reveals the link between ATM activation and its exchange. Cancer research, 81(2), 426-437.

Publication 2020

FACS Calibur flow cytometer APC-TCRβ (clone H57-597, PE-IgM anti-CD43 magnetic beads, anti-CD40 FITC-conjugated IgG1 PECy5-conjugated B220

Antibodies Cd43 Cells Clone Fitc Hematopoietic Igg1 Lymphocyte Mice Serum Splenic Supplements Tcrβ

Corresponding Organization : Cancer Genetics (United States)

Other organizations : The University of Texas at Austin, St. Jude Children's Research Hospital, University of California, San Diego

Top 5 similar protocols

Variable analysis

independent variables

Mouse age (5–8 weeks)

dependent variables

Lymphocyte development
In vitro class switch recombination (CSR)

control variables

Antibodies used for flow cytometry: PE-CD4, FITC-CD8α, APC-TCRβ, PE/Cy7 TER-119, FITC-CD43, PE-Cy5-B220, PE-IgM
In vitro CSR culture conditions: CD43- splenic cells, RPMI media, serum supplements, IL-4 (20 ng/mL), anti-CD40 (1 μg/mL), FITC-conjugated IgG1, PE-Cy5-conjugated B220

positive controls

Previous studies (references 10, 31, 32) for lymphocyte development and in vitro CSR protocols

negative controls

No negative controls explicitly 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!